Regulation and control of RNA on expression of SUV39H1

Abstract

The present invention provides compositions and methods for modulating the expression of SUV39H1 using an inhibitory or activating polynucleotide based on a long non-coding RNA or short hairpin RNA (shRNA) sequence.

Description

Technical Field

[0001] The present invention relates to compositions and methods for regulating SUV39H1 expression using RNA. Background Art

[0002] Long non-coding RNAs (lncRNAs) are regulators of gene expression and are involved in many cellular processes. Their expression has been found to be particularly tissue-specific or development-stage-specific (Ahmad et al., 2021).

[0003] Small hairpin RNAs (shRNAs) are RNA sequences, typically about 80 base pairs in length, which include an internal hybridization region that produces a hairpin structure. shRNA molecules are processed intracellularly to form siRNAs, which in turn knockdown gene expression. The advantage of shRNAs is that they can be incorporated into plasmid vectors and integrated into genomic DNA to achieve long-term or stable expression, thereby knockdown target mRNA for a long time.

[0004] Histone methyltransferases promote chromatin rearrangement into euchromatin (open, transcriptionally active chromatin) and heterochromatin (closed, inactive chromatin) by post-translationally modifying histone molecules at the nucleosome. Histone methyltransferases actively control gene expression and cell fate. SUV39H1 is one of the first methyltransferases to be identified (Aagaard et al., 1999). It has been shown to be involved in many developmental processes, particularly in the development of the immune system (Rao et al., 2017; Nicetto and Zaret, 2019; Allan et al., 2012; Pace et al., 2018).

[0005] Pace et al., 2018 and International Patent Publication WO 2018 / 234370 disclose that inhibiting the expression of SUV39H1 in T cells and NK cells enhances their memory potential and improves their survival ability. Summary of the Invention

[0006] The present disclosure provides compositions and methods for regulating SUV39H1 expression using RNA.

[0007] The present invention relates to a cell comprising a first nucleic acid, which is a heterologous nucleic acid expressing RNA, comprising the nucleobase sequence of [SEQ ID NO: 1] (lncRNA AF196970.3) or a fragment thereof capable of inhibiting the expression of SUV39H1 in the cell; a heterologous nucleic acid comprising the nucleotide sequence of [SEQ ID NO: 5] (genomic sequence encoding AF196970.3 (ENSG00000232828)) or a fragment thereof expressing RNA capable of inhibiting the expression of SUV39H1 in the cell; a nucleic acid expressing RNA, comprising the nucleobase sequence of [SEQ ID NO: 1] (lncRNA AF196970.3) or a fragment thereof capable of inhibiting the expression of SUV39H1 in the cell, said nucleic acid being operably linked to a heterologous expression control sequence (such as a promoter, enhancer, other regulatory sequences); or a nucleic acid operably linked to a heterologous expression control sequence, comprising the nucleotide sequence of [SEQ ID NO: 5] (genomic sequence encoding AF196970.3 (ENSG00000232828)) or a fragment thereof expressing RNA capable of inhibiting the expression of SUV39H1 in the cell, said nucleic acid being operably linked to a heterologous expression control sequence. The first nucleic acid may comprise the nucleotide sequence of any one of SEQ ID NOs: 5-9 or a fragment thereof. The first nucleic acid may express RNA comprising the nucleobase sequence of [SEQ ID NO: 2] (exon 1 of lncRNA AF196970.3) or a fragment thereof capable of inhibiting the expression of SUV39H1 in the cell. The first nucleic acid may express RNA comprising the nucleobase sequence of [SEQ ID NO: 3] (exon 2 of lncRNA AF196970.3) or a fragment thereof capable of inhibiting the expression of SUV39H1 in the cell. The first nucleic acid may express RNA comprising the nucleobase sequence of [SEQ ID NO: 4] (exon 3 of lncRNA AF196970.3) or a fragment thereof capable of inhibiting the expression of SUV39H1 in the cell. In any of the foregoing embodiments, the first nucleic acid may express RNA having a length of at least about 12-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 250-750, 500-750 or more bases.

[0008] The present invention also relates to a cell comprising a nucleic acid, which is a heterologous nucleic acid, expressing an RNA, comprising the nucleotide sequence of any one of SEQ ID NO: 13-17 or SEQ ID NO: 26-30 or a fragment or derivative thereof capable of inhibiting SUV39H1 expression in the cell; or a fragment of an RNA-expressing nucleic acid capable of inhibiting SUV39H1 expression in the cell; an RNA-expressing nucleic acid comprising the nucleotide sequence of any one of SEQ ID NO: 13-17 or SEQ ID NO: 26-30 or a fragment thereof capable of inhibiting SUV39H1 expression in the cell, said nucleic acid being generally operably linked to a heterologous expression control sequence (e.g., a promoter, an enhancer, other regulatory sequences). The nucleic acid may comprise the nucleotide sequence of any one of SEQ ID NO: 13-17 or 26-30 or a fragment thereof. The present invention also relates to a cell comprising a heterologous polynucleotide, which heterologous polynucleotide comprises the nucleotide sequence of any one of SEQ ID NO: 32-36 and 45-49 or a fragment thereof capable of inhibiting SUV39H1 expression in the cell. In some embodiments, the nucleic acid may be operably linked to a heterologous expression control sequence (e.g., a promoter, an enhancer, other regulatory sequences).

[0009] The cell may be a modified immune cell.

[0010] The cell may be any of the following cell types: T cell, CD4+ T cell, CD8+ T cell, CD4+ and CD8+ T cells, NK cell, Treg cell, Tm cell, memory stem T cell (TSCM), TCM cell, TEM cell, monocyte, dendritic cell, macrophage, T cell progenitor, NK cell progenitor, pluripotent stem cell, induced pluripotent stem cell (iPSC), hematopoietic stem cell (HSC), adipose stem cell (ADSC), myeloid or lymphoid pluripotent stem cell.

[0011] The cell may comprise one or more engineered receptors, two or more, or three or more.

[0012] The cell may contain a second heterologous nucleic acid that expresses one or more engineered receptors. The engineered receptor may comprise: a) an extracellular antigen-binding domain that specifically binds an antigen, optionally comprising an antibody heavy chain variable region and / or an antibody light chain variable region, and may optionally be bispecific or trispecific; b) a transmembrane domain, optionally comprising a transmembrane domain fragment of the α, β, or ζ chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD34, CD137, or CD154, NKG2D, OX40, ICOS, 2B4, DAP10, DAP12, CD40; c) one or more co-stimulatory domains optionally from 4-1BB, CD28, ICOS, OX40, DAP10, or DAP12, 2B4, CD40, FCER1G; and d) an intracellular signaling domain comprising an intracellular signaling domain or any fragment thereof from CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CDS, CD22, CD79a, CD79b, or CD66d, 2B4. The engineered receptor may be a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain (optionally an scFv), a transmembrane domain (optionally from CD28, CD8, or CD3ζ), one or more co-stimulatory domains (optionally from 4-1BB, CD28, ICOS, OX40, or DAP10), and an intracellular signaling domain from CD3ζ, optionally wherein ITAM2 and ITAM3 have been inactivated.

[0013] The engineered receptor may also be a modified TCR.

[0014] The modified TCR can comprise (a) an extracellular domain comprising an antigen-binding fragment of an antibody or CDRs, preferably all three CDRs of the heavy-chain variable region (VH) and / or the light-chain variable region (VL), and (b) a native or variant constant region of an α, β, γ or δ chain. For example, the modified TCR can comprise one or more heterologous polypeptides, such as (a) a VH of an antibody fused to TRBC1 (usually human or murine TRBCl) or TRBC2 (usually human or murine TRBC2) or a fragment or variant thereof having at least 90% sequence identity thereto, or a fragment or variant of TRBC1 or TRBC2 having at least 90% sequence identity thereto, and (b) a VL of an antibody fused to TRAC (human or murine TRAC sequence) or a fragment or variant thereof having at least 90% sequence identity thereto, or a fragment or variant of TRAC having at least 90% sequence identity thereto. The modified TCR can also optionally comprise a native or variant CD3ζ polypeptide, such as a modified CD3ζ polypeptide in which one or two ITAM domains (e.g., ITAM2 and ITAM3) have been deleted.

[0015] Recombinant HLA-independent (or non-HLA-restricted) modified TCRs (referred to as "HI-TCRs") that bind to an antigen of interest in an HLA-independent manner are typically described in International Application WO 2019 / 157454. Such HI-TCRs comprise an antigen-binding chain that comprises: (a) a heterologous antigen-binding domain that binds to an antigen in an HLA-independent manner, such as an antigen-binding fragment of an immunoglobulin variable region; and (b) a constant domain capable of associating with (and thus activating) a CD3ζ polypeptide. Preferably, the antigen-binding domain or a fragment thereof comprises: (i) the heavy-chain variable region (VH) of an antibody and / or (ii) the light-chain variable region (VL) of an antibody. The constant domain of the TCR is, for example, a native or modified TRAC polypeptide (usually a human or murine TRAC sequence), or a native or modified TRBC polypeptide (usually a human or murine TRBC sequence). The constant domain of the TCR is, for example, a (human or murine) native TCR constant domain (α or β) or a fragment thereof. Unlike chimeric antigen receptors, which typically themselves comprise an intracellular signaling domain, HI-TCRs do not directly generate an activation signal; rather, the antigen-binding chain associates with a CD3ζ polypeptide and thus activates the CD3ζ polypeptide. Immune cells comprising a recombinant TCR provide excellent activity when the density of the antigen on the cell surface is low (usually median density), less than about 10,000 molecules per cell, such as less than about 5,000, 4,000, 3,000, 2,000, 1,000, 500, 250 or 100 molecules per cell.

[0016] Thus, an engineered receptor can typically be a modified TCR comprising a first antigen-binding chain and a second antigen-binding chain, wherein the first antigen-binding chain comprises an antigen-binding fragment of an antibody heavy chain variable region (VH), and the second antigen-binding chain comprises an antigen-binding fragment of an antibody light chain variable region (VL); wherein each of the first antigen-binding chain and the second antigen-binding chain comprises a TRAC polypeptide or a TRBC polypeptide, optionally wherein at least one of the TRAC polypeptide and the TRBC polypeptide is endogenous, and optionally wherein one or both of the endogenous TRAC polypeptide and the TRBC polypeptide are inactivated.

[0017] The cell can also comprise a chimeric co-stimulatory receptor comprising (a) an extracellular and transmembrane domain of CD86, 41BBL, CD275, CD40L, OX40L, PD-1, TIGIT, 2B4 or NRP1, or a fragment or variant thereof, and (b) an intracellular co-stimulatory molecule of CD28, 4-1BB, 0X40, ICOS, CD27, CD40 or CD2, or a fragment or variant thereof. In some embodiments, the chimeric co-stimulatory receptor comprises (a) an extracellular domain of a co-stimulatory ligand, optionally from CD80, (b) a transmembrane domain, optionally from CD80, and (c) an intracellular domain of a co-stimulatory molecule, optionally CD28, 4-1BB, OX40, ICOS, DAP10, CD27, CD40, NKGD2 or CD2, preferably 4-1BB.

[0018] The extracellular antigen-binding domain of the cell can bind an antigen with a KD affinity of about 1 x 10 -7 or lower, about 5 x 10 -8 or lower, about 1 x10 -8 or lower, about 5 x 10 -9 or lower, about 1 x 10 -9 or lower, about 5 x 10 -10 or lower, about 1 x 10 -10 or lower, about 5 x 10 -11 or lower, about 1 x 10 -11 or lower, about 5 x 10 -12 or lower or about 1 x 10 -12 or lower (the lower the number, the higher the affinity) to bind the antigen. The density of the antigen bound by the extracellular antigen-binding domain on the cell surface can be low, less than about 10,000 molecules per cell, or less than about 5,000 molecules, or less than about 2,000 molecules.

[0019] The extracellular antigen-binding domain can bind to an antigen, for example,

[0020] Orphan tyrosine kinase receptors ROR1, tEGFR, Her2, p95HER2, LI-CAM, CD19, CD20, CD22, mesothelin, CEA, Claudin 18.2, hepatitis B surface antigen, folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, CD70, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, ErbB3 or ErbB4, FBP, FcRH5, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-α, IL-13R-α2, kdr, K light chain, BCMA, Lewis Y, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D ligand, NY-ESO-1, MART-1, gp100, oncofetal antigen, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate specific antigen (PSMA), estrogen receptor, progesterone receptor, ephrinB2, CD 123, CS-1, c-Met, GD-2, MAGE A3, CE7 or Wilms tumor 1 (WT-1). In some embodiments, the antigen can be any neoantigen peptide disclosed in International Patent Publications WO 2021 / 043804, WO 2022 / 189620, WO 2022 / 189626, WO 2022 / 189636, the entire contents of which are incorporated herein by reference.

[0021] The cell can comprise two engineered antigen receptors, each receptor binding a different antigen.

[0022] The cell can be autologous or allogeneic.

[0023] Expression of SUV39H1 in the cell can be reduced or inhibited by at least about 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95%.

[0024] The cell can initially be isolated from a subject having cancer or at risk of developing cancer.

[0025] The present invention also relates to modified oligonucleotides comprising a nucleobase sequence of at least about 12 bases in length from any one of [SEQ ID NO: 1-4], wherein the modified oligonucleotides comprise one or more of a modified backbone linkage, a modified sugar moiety, a modified phosphate moiety, a modified nucleobase, or a chemical conjugate moiety. The length of the modified oligonucleotides can be at least about 12-50, 50-75, or 50-100 bases. The modified oligonucleotides can comprise a modified backbone linkage comprising a phosphorothioate, phosphonoacetate, thiophosphonoacetate, methylphosphonate, boranophosphate, or dithiophosphate moiety. The modified oligonucleotides can comprise a modified sugar that is modified to replace the 2'-OH group with another group, which another group is optionally H, -OR, -R (wherein R can be, for example, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar), halogen, -F, -Br, -Cl, or -I, -SH, -SR (wherein R can be, for example, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar), -arabinose, F-arabinose, amino (wherein the amino can be, for example, NH2; alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); or cyano (-CN); optionally 2'-O-methoxy, 2'-O-methoxyethyl modification, 2'-fluoro, 2'-deoxy, or a combination thereof. The modified oligonucleotides can comprise a modified nucleobase that comprises one or more 5-methylcytosine, modified uridines such as 5-(2-amino)propyl uridine and 5-bromouridine, modified adenosines and guanosines such as modified at position 8, such as 8-bromoguanosine, deazapurines such as 7-deazaadenosine, or O- and N-alkylated nucleotides such as N6-methyladenosine, or polycyclic modified nucleotides (e.g., tricyclic; and "unlocked" forms such as glycol nucleic acid (GNA) (e.g., R-GNA or S-GNA, wherein ribose is replaced by an ethylene glycol unit in the phosphodiester bond), or threose nucleic acid (TNA, wherein ribose is replaced by α-L-threofuranosyl-(3'→2'))).

[0026] The present invention also relates to a nucleic acid operably linked to a heterologous expression control sequence, said nucleic acid comprising (a) a nucleotide sequence encoding or expressing an RNA, which comprises the nucleobase sequence of [SEQ ID NO: 1] (lncRNA AF196970.3) or the nucleotide sequence of any one of SEQ ID NOs: 13-17 and 26-30 or a fragment thereof capable of inhibiting the expression of SUV39H1 in cells, or (b) the nucleotide sequence of [SEQ ID NO: 5] (the genomic sequence encoding AF196970.3 (ENSG00000232828)) or the nucleotide sequence of any one of SEQ ID NOs: 32-36 and 45-49 or a fragment thereof expressing an RNA capable of inhibiting the expression of SUV39H1 in cells. The nucleic acid may comprise the nucleotide sequence of any one of SEQ ID NOs: 5-9, 32-36, 42-49 or a fragment or fragments thereof. The heterologous control sequence may be a constitutive, inducible or tissue-specific promoter, optionally EF1α, CMV, SFFV, hPGK, RPBSA or CAG.

[0027] The present invention also relates to a vector comprising the nucleic acid described herein and one or more additional expression control sequences. The vector is a viral vector, optionally an adenovirus, adeno-associated virus (AAV), poxvirus, papillomavirus, lentivirus, retrovirus, herpesvirus, foamy virus or Semliki Forest virus vector, and includes pseudotyped viruses.

[0028] The present invention also relates to a delivery vehicle, optionally a liposome, lipid-containing complex, nanoparticle, gold particle or polymeric complex comprising the above nucleic acid or vector.

[0029] The present invention also relates to a method of preparing the cells described herein, comprising at least the following steps: (a) introducing (i) the nucleic acid described herein or (ii) the vector described herein into the cells, and optionally (b) introducing a nucleic acid encoding an antigen-specific receptor into the cells.

[0030] The present invention also relates to a method for preparing the cells described herein, comprising at least the following steps: introducing a heterologous expression control sequence into a cell in such a way that it is operably linked to an endogenous nucleic acid that expresses RNA, the endogenous nucleic acid comprising the nucleobase sequence of [SEQ ID NO: 1] (lncRNA AF196970.3), or the nucleotide sequence of any one of SEQ ID NOs: 13-17, 26-30 or a fragment thereof that is capable of inhibiting the expression of SUV39H1 in the cell, optionally a nucleic acid comprising the nucleotide sequence of [SEQ ID NO: 5] (genomic sequence encoding AF196970.3 (ENSG00000232828) or an allelic variant thereof) or the nucleotide sequence of any one of SEQ ID NOs: 32-36 and 45-49.

[0031] The present invention also relates to a method for treating a disease using the cells described herein, comprising administering to a subject in need thereof an effective amount of the cells for treating the disease, optionally cancer, an infectious disease, an autoimmune disease, an inflammatory disease or an allergic disease, wherein the cells express one or more antigen-specific receptors that bind to an antigen associated with the disease.

[0032] The present invention also relates to a method for treating a subject suffering from cancer, comprising administering to the subject: (1) the cells described herein; and (2) a second cancer therapeutic agent. The second cancer therapeutic agent can be an immune checkpoint modulator, a cancer vaccine, a chemotherapeutic agent or an anti-angiogenic agent.

[0033] The present invention also relates to a method for treating a subject suffering from cancer, comprising administering to the subject: (1) the cells described herein, wherein the cells are T cells, NK cells or T cell progenitors that comprise a genetically engineered antigen receptor, wherein the expression of the SUV39H1 gene is inhibited, and wherein the inhibition of the SUV39H1 gene results in enhanced anti-cancer activity of the immune cells; and (2) an immune checkpoint modulator. The immune checkpoint modulator can be an inhibitor of PD1, CTLA4, LAG3, BTLA, OX2R, TIM-3, TIGIT, LAIR-1, PGE2 receptor, EP2 / 4 adenosine receptor or A2AR. The immune checkpoint modulator can be an anti-PD-1 inhibitor or an anti-PDL-1 inhibitor. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Shows the SUV39H1 locus containing the gene ENSG00000232828 encoding (expressing) lncRNA AF196970.3 in the UCSC Genome Browser (genome-euro.ucsc.edu).

[0035] Figure 2AShows lncRNA AF196970.3 and its three exons, Figure 2B Shows the overlap in the genomic region and the shared nucleotides between exon 2 of lncRNA AF196970.3 and exon 3 of SUV39H1, and Figure 2C Shows the overlap in the genomic region and the shared nucleotides between exon 3 of lncRNA AF196970.3 and exon 2 of SUV39H1.

[0036] Figure 3A Shows the tissue gene expression of AF196970.3 in a large number of tissues, Figure 3B Shows the tissue gene expression of SUV39H1 in a large number of tissues, Figure 3C Shows the single-cell expression of AF196970.3 in different tissues, Figure 3D Shows the single-cell expression of SUV39H1 in different tissues, and Figure 3E Shows the expression of transcript ENST00000416061.1 of AF196970.3 in different tissues.

[0037] Figure 4A 、 Figure 4B and Figure 4C Show the expression levels of AF196970.3 and SUV39H1 in single-cell RNAseq-derived clusters from glioma, head and neck squamous cell carcinoma, and hepatocellular carcinoma, respectively.

[0038] Figure 5 Shows a plasmid map representing the Piggy Bac backbone with a GFP-puromycin reporter gene, including the exon sequence of lncRNA SUV39H1 with a CMV promoter (5A), or the exon sequence of lncRNA SUV39H1 with an hPKG promoter (5B).

[0039] Figure 6 A shows a schematic diagram of the experimental procedure for Piggy Bac transfection in HEK293 FT cells using Figure 5 two plasmid constructs. Figure 6 B shows the GFP expression levels measured by flow cytometry, and the comparison between untransfected cells and cells transfected with either the empty Piggy Bac construct or one of the lncRNA SUV39H1 Piggy Bac plasmids. Figure 6 C shows the expression of SUV39H1 and actin in cells by Western blot. Quantification of the SUV39H1 protein level normalized to actin. Figure 6 D shows a schematic diagram of the experimental procedure for the expression of lncRNA SUV39H1 in CD8+ T cells.

[0040] Figure 7 A shows a schematic diagram of the shRNA target sequence on the SUV39H1 gene. Figure 7 B shows the target sequences and loop sequences of different shRNAs targeting SUV39H1. Figure 7 C shows a plasmid map of a lentiviral construct for expressing shRNA in cells using the U6 promoter and the EGFP reporter gene.

[0041] Figure 8 A shows the experimental procedure for the expression of shRNA in HEK293 FT cells. Figure 8 B shows the GFP expression levels measured by flow cytometry: comparison of untransduced cells with cells transduced with scrambled shRNA or one of five shRNAs targeting SUV39H1. Figure 8 C shows the expression of SUV39H1 and actin in cells by Western blot.

[0042] Figure 9 A shows the experimental procedure for the expression of shRNA in CD8+ T cells. Figure 9 B shows the GFP expression levels measured by flow cytometry: comparison of untransduced cells with cells transduced with scrambled shRNA or one of five shRNAs targeting SUV39H1. Shown are the results of a representative donor. Figure 9 C shows the expression of SUV39H1 and actin in cells by Western blot. Control cells in the absence (mock) or presence (gRNA SUV) of gRNA targeting SUV39H1. Figure 9 D shows the level of trimethylation of H3K9 (geometric mean fluorescence intensity) in T cells measured by flow cytometry. Shown are the results of a representative donor. Figure 9 E- Figure 9 F shows the expression of the memory marker CD27 in T cells on day 7 measured by flow cytometry: comparison of the scrambled sequence with shRNA 1 for 3 donors (replicate means). Figure 9 F shows the expression of the memory marker CD27 in T cells on day 14 measured by flow cytometry: comparison of the scrambled sequence with shRNA 1 for 2 donors (replicate means). Detailed Description

[0043] SUV39H1 is an H3K9-histone methyltransferase that plays a role in silencing memory and stem cell programs during the terminal differentiation of effector CD8+ T cells. Conversely, silencing of SUV39H1 has been shown to enhance long-term memory potential and improve viability. Human SUV39H1, see UniProt accession number O43463.

[0044] The present disclosure relates to the identification of an antisense lncRNA, AF196970.3, which has a silencing function on SUV39H1 in human cells, inhibits the expression of SUV39H1, thereby reducing the level of SUV39H1 protein in the cells. The expression pattern of AF196970.3 is very similar to that of SUV39H1. It is detected in many different cell types, for example, at the highest levels in endothelial cells, fibroblasts, and myocytes, similar to SUV39H1. AF196970.3 is expressed in human tumor infiltrating lymphocytes of at least three different types of cancer, including: glioma, head and neck squamous cell carcinoma, and hepatocellular carcinoma. The expression level of AF196970.3 is correlated with the level of SUV39H1 and, like SUV39H1, is higher in proliferating T cells.

[0045] The present disclosure also relates to the identification of short hairpin RNAs that comprise any one of the target sequences set forth in SEQ ID NOs: 13-17 (shRNA1-5 described herein), or that comprise any one of the sequences SEQ ID NOs: 26-30 (comprising a target sequence, a loop sequence, and a guide sequence to form an shRNA).

[0046] The present disclosure provides inhibitory polynucleotides and their uses. The expression of SUV39H1 in cells can be inhibited by increasing the ectopic or endogenous expression of the antisense lncRNA sequence, or shRNA or similar polynucleotides based on the sequence, its variants and / or fragments, as disclosed herein. The reduction of SUV39H1 levels in cells affects the commitment of cells to terminal differentiation and / or prolongs the survival time. The lncRNA sequence or its fragment or variant includes a shorter RNA or a similar polynucleotide based on the lncRNA sequence, and the shRNA described herein, which generally includes one of the target sequences SEQ ID NOs: 13-17, or any of SEQ ID NOs: 26-30, which can act as a local inhibitor of SUV39H1 transcription and / or act through its overlapping antisense exons. In any of these embodiments, the inhibition of SUV39H1 reduces the expression and / or activity of SUV39H1 by at least about 20%, 30%, 40%, 50%, 60%, 70%, 75% or 80% or more compared to wild-type cells in which SUV39H1 expression is not regulated. In some embodiments, the RNA inhibitors described herein reduce H3K9 trimethylation by at least about 20%, 30%, 40%, 50%, 60%, 70%, 75% or 80% or more compared to wild-type cells in which SUV39H1 expression is not regulated (see also the results section of the Examples assay).

[0047] The present disclosure also provides activating polynucleotides and their uses. The expression of SUV39H1 in cells is increased by inhibiting the expression of the lncRNA sequence AF196970.3 or increasing its degradation. RNA interference (RNAi), short hairpin RNA (shRNA) and antisense oligonucleotides (ASO) can be used to inhibit the expression of RNA or reduce its degradation.

[0048] Immune cells, particularly T cells or NK cells, in which the expression of SUV39H1 is inhibited, can exhibit an enhanced central memory phenotype, increased survival and persistence after adoptive transfer, and reduced exhaustion. In particular, such cells accumulate and are reprogrammed more efficiently into long-lived central memory T cells. Such cells induce tumor cell rejection more efficiently and show enhanced cancer therapeutic efficacy. Definition

[0049] As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

[0050] Likewise, as used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the absence of a combination when interpreted in the alternative ("or").

[0051] As used herein, the term "about", when referring to a measurable value (such as the amount of a polypeptide, dose, time, temperature, enzyme activity, or other biological activity, etc.), is intended to cover variations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount.

[0052] The term "antibody" as used herein is used in the broadest sense and includes polyclonal antibodies and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab’ fragments, Fv fragments, recombinant IgG (rIgG) fragments, variable heavy chain (VH) regions capable of specifically binding an antigen, single-chain antibody fragments, including single-chain variable fragments (scFv) and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses recombinant and / or otherwise modified immunoglobulin forms, such as intracellular antibodies, peptide antibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific (e.g., bispecific) antibodies, diabodies, triabodies, and tetra-bodies, tandem bis-scFv, tandem tris-scFv. Unless otherwise specified, the term "antibody" shall be understood to cover its functional antibody fragments. The term also covers intact or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgG1, IgG2, IgG3, IgG4, IgM, IgE, IgA, and IgD. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region.

[0053] "Antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab’, Fab’-SH, F(ab’)2; diabodies; linear antibodies; variable heavy chain (VH) regions, VHH antibodies, single-chain antibody molecules such as scFv and single-domain VH single antibodies; and multispecific antibodies formed from antibody fragments. In certain embodiments, the antibody is a single-chain antibody fragment comprising a variable heavy chain region and / or a variable light chain region, such as scFv.

[0054] "Inactivation" or "disruption" of a gene refers to an alteration in the genomic DNA sequence that results in reduced or absent expression of the gene, or in the expression of a non-functional gene product. Exemplary methods include gene silencing, knockdown, knockout, and / or gene disruption techniques, such as gene editing by inducing breaks and / or homologous recombination. Examples of such gene disruptions are insertions, frameshift and missense mutations, deletions, knock-ins, and knockouts of a gene or a portion of a gene, including deletion of an entire gene. Such disruptions can occur in the coding region, e.g., in one or more exons, resulting in the inability to produce a full-length product, a functional product, or any product, e.g., by insertion of a stop codon. Such disruptions can also occur due to disruption of a promoter, enhancer, or other region that affects transcriptional activation, thereby preventing transcription of the gene. Gene disruption includes gene targeting, including targeted gene inactivation by homologous recombination.

[0055] As used herein, "inhibition" of a gene product refers to a reduction in its activity and / or gene expression by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more compared to the activity or expression level of the wild-type that is not inhibited or repressed.

[0056] As used herein, "express" or "expression" means that a gene sequence is transcribed and optionally translated. If a gene expresses a non-coding RNA, expression generally results in an RNA after transcription and optional splicing. If the gene is a coding sequence, expression generally results in a polypeptide after transcription and translation.

[0057] As used herein, "expression control sequence" means a nucleotide sequence that affects the transcription, RNA processing, RNA stability, or translation of a relevant nucleotide sequence. Examples include, but are not limited to, promoters, enhancers, introns, translation leader sequences, polyadenylation signal sequences, transcription initiators, and transcription and / or translation termination regions (i.e., termination regions).

[0058] As used herein, "fragment" means a portion of a reference sequence (polynucleotide or polypeptide) that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the length of the full-length sequence.

[0059] As used herein, "heterologous" refers to a polynucleotide or polypeptide that contains sequences that do not have the same relationship to each other in nature. For example, a heterologous sequence either originates from another species or from the same species or organism but has been modified from its original form or the form in which it is predominantly expressed in a cell. Thus, a heterologous polynucleotide includes a nucleotide sequence that is derived from and inserted into the same native cell type, but the nucleotide sequence is present in a non-natural state, e.g., different copy numbers, and / or is controlled by regulatory sequences different from those found in nature, and / or is located at a different position (adjacent to a different nucleotide sequence) from its original position.

[0060] As used herein, the terms "nucleic acid", "nucleotide sequence", and "oligonucleotide" or "polynucleotide" are used interchangeably and encompass RNA and DNA, including cDNA, genomic DNA, mRNA, synthetic (e.g., chemically synthesized) DNA or RNA, and chimeras of RNA and DNA. The term "polynucleotide", "nucleotide sequence", or "nucleic acid" refers to a chain of nucleotides regardless of chain length. The nucleic acid can be double-stranded or single-stranded. If single-stranded, the nucleic acid can be the sense strand or the antisense strand. Nucleic acids can be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids with altered base pairing capabilities or increased resistance to nucleases. The present disclosure also provides a nucleic acid that is complementary (which can be fully complementary or partially complementary) to the nucleic acid, nucleotide sequence, or polynucleotide described herein. Modified bases (modified nucleobases), such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine, etc. can also be used for antisense, dsRNA, and ribozyme pairing. For example, polynucleotides containing C-5 propyne analogs of uridine and cytidine have been shown to bind RNA with high affinity and are potent antisense inhibitors of gene expression. Other modifications can also be made, such as modifications to the phosphodiester backbone or the 2'-hydroxyl group in the RNA ribose group.

[0061] As used herein, "operably linked" means that an element (e.g., an expression control sequence) is configured to perform its normal function on a nucleotide sequence of interest. For example, a promoter operably linked to a nucleotide sequence of interest is capable of effecting the expression of the nucleotide sequence of interest. The expression control sequence need not be adjacent to the nucleotide sequence of interest so long as they are capable of directing its expression.

[0062] As used herein, the term "percent identity" between two sequences refers to the percentage of identical bases or amino acids between the sequences obtained by aligning the two sequences to be compared optimally. This percentage is purely statistical, and the differences between the two sequences are randomly distributed across the two sequences. Bases are considered complementary if they hybridize under normal conditions. For example, modified nucleobases can be aligned in a manner similar to the bases that they mimic in hybridization patterns. As used herein, "optimal alignment" or "optimum alignment" refers to the alignment that gives the highest percent identity (see above). Sequence comparison between two nucleic acid sequences (also referred to herein as nucleotide sequences or nucleobase sequences) is typically achieved by comparing these sequences that have been previously aligned optimally. Such comparison is achieved over comparison segments to identify and compare local regions of similarity. In addition to manually, the optimal sequence alignment for comparison can be achieved by using the global homology algorithm developed by SMITH and WATERMAN (Advances in Applied Mathematics (Ad. App. Math), Vol. 2, p. 482, 1981), by using the local homology algorithm developed by NEEDLEMAN and WUNSCH (Journal of Molecular Biology (J. Mol. Biol), Vol. 48, p. 443, 1970), by using the similarity method developed by PEARSON and LIPMAN (Proceedings of the National Academy of Sciences of the United States of America (Proc. Natl. Acad. Sci. USA), Vol. 85, p. 2444, 1988), by using computer software that employs such algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA, TFASTA in the Wisconsin Genetics software package, Genetics Computer Group, 575 Science Dr., Madison, Wisconsin, USA), by using the MUSCLE multiple alignment algorithm (Edgar, Robert C, Nucleic Acids Research, Vol. 32, p. 1792, 2004). To obtain an optimal local alignment, the BLAST software can preferably be used. The percent identity between two sequences is determined by comparing the two optimally aligned sequences, and the sequences can include additions or deletions relative to the reference sequence in order to obtain the optimal alignment between the two sequences. The percent identity is calculated by determining the number of positions that are identical between the two sequences, then dividing that number by the total number of positions compared, and then multiplying the resulting value by 100 to obtain the percent identity between the two sequences.

[0063] As used herein, "treatment" or "treating" involves applying the cells of the present disclosure or a composition comprising the cells to a patient in need thereof, with the aim of curing, healing, alleviating, mitigating, altering, remedying, reducing, improving, or affecting a disease (such as cancer) or any symptom of a disease (such as cancer). In particular, the term "treatment" refers to reducing or alleviating at least one adverse clinical symptom associated with a disease. With regard to cancer treatment, the term "treatment" also refers to slowing or reversing the progression of uncontrolled cell proliferation of a tumor, i.e., shrinking an existing tumor and / or preventing tumor growth. The term "treatment" also refers to inducing apoptosis of cancer or tumor cells in a subject.

[0064] As used herein, a "variant" refers to a sequence (polynucleotide or polypeptide) having a mutation (deletion, substitution, or insertion) that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a reference sequence over its full length or over a region of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or 1100 nucleotides or amino acids. With regard to polynucleotide sequences, variants also encompass polynucleotides that hybridize to a reference sequence or its complement under stringent conditions.

[0065] As used herein, a "vector" is any nucleic acid molecule used for transferring or expressing nucleic acids in cells. The term "vector" includes viral and non-viral (e.g., plasmid) nucleic acid molecules for introducing nucleic acids into cells in vitro, ex vivo, and / or in vivo. A vector may include expression control sequences, restriction sites, and / or selectable markers. A "recombinant" vector refers to a vector containing one or more heterologous nucleotide sequences. Inhibitory polynucleotide that inhibits SUV39H1 expression

[0066] The locus of SUV39H1 is located on the X chromosome (position p11.23, 48695554 - 48709016 in the GRCh38.p13 assembly). At the same locus, in an antiparallel orientation, the unannotated gene ENSG00000232828 is located at positions 48698963 - 48737163 ( Figure 1 ). Both genes have annotated promoter regions.

[0067] AF196970.3 (SEQ ID NO: 1) is a predicted RNA sequence expressed after transcription and splicing of ENSG00000232828 (SEQ ID NO: 5). SEQ ID NO: 1 is a 925-base sequence containing three exons ( Figure 2A ). AF196970.3 exon 1 (SEQ ID NO: 2) is 125 bases in length and has no significant complementarity with the SUV39H1 gene. AF196970.3 exon 2 (SEQ ID NO: 3) is 600 bases in length and is mostly antiparallel to exon 3 of the SUV39H1 gene with 100% similarity ( Figure 2B ). AF196970.3 exon 3 (SEQ ID NO: 4) is 200 bases in length and is antiparallel to a part of exon 2 of the SUV39H1 gene (42.5% similarity) and a part of the adjacent intron ( Figure 2C ).

[0068] The inhibitory polynucleotides of the present disclosure for the cells and methods of the present disclosure include AF196970.3 (SEQ ID NO: 1) or fragments or variants thereof. Such fragments include fragments of any of SEQ ID NOs: 1-4, having a length of about 12-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 250-750, 500-750 bases or more. Variants include polynucleotides, or chemically modified polynucleotides, in which SEQ ID NO: 1 or the aforementioned fragments of SEQ ID NO: 1 have been mutated, including deletions, substitutions, modifications (including chemical modifications) or insertions. Such variants may have a nuclear base sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any of SEQ ID NOs: 1-4 or any fragment thereof (e.g., a fragment of the aforementioned length of SEQ ID NO: 1). Variants include, for example, any of the aforementioned fragments of SEQ ID NO: 1 that are linked to additional nucleotides at the 5' end or 3' end or both. Variants also include RNAs produced by alternative splicing of ENSG00000232828 (SEQ ID NO: 5) or RNAs produced by allelic variants of ENSG00000232828 (SEQ ID NO: 5).

[0069] The inhibitory RNAs according to the present disclosure also include the shRNAs described herein. The principles and design of shRNAs are described, for example, in Moore CB, Guthrie EH, Huang MT, Taxman DJ, Short hairpin RNAs (shRNAs): Design, delivery, and evaluation for gene knock-down, Methods Mol Biol 2010; 629: 141-58, but see also Taxman DJ, siRNA and shRNA design. In: Helliwell TDC, editor. RNA Interference Methods for Plants and Animals. Vol. 10. CABI; Oxfordshire, UK: 2009, pp. 228-253 (Helliwell TDC, editor. RNA Interference Methods for Plants and Animals. Vol. 10. CABI; Oxfordshire, UK: 2009. pp. 228-253). The mechanism of RNAi is based on sequence-specific degradation of host mRNA by cytoplasmic delivery of double-stranded RNA (dsRNA) identical to the target sequence. shRNAs can be transfected as plasmid vectors encoding shRNAs transcribed by a heterologous promoter, but can also be delivered into mammalian cells by infecting cells with virus-generated vectors. shRNAs are capable of DNA integration and typically consist of two complementary 19bp-22bp RNA sequences linked by a short loop of 4nt-11nt, similar to the hairpin in naturally occurring miRNAs. Example loop sequences are also described herein, but any variant based on the classical knowledge of those skilled in the art can be used (see also the references on shRNA design above). Generally, shRNAs contain or are encoded by a nucleotide sequence that contains one of the sequences of SEQ ID NOs: 13-17 and 26-30 and SEQ ID NOs: 32-36 and 45-49, respectively).

[0070] Chemical modifications include one or more of modified backbone linkages, modified sugar moieties, modified phosphate moieties, modified nucleobases, or chemical conjugate moieties. Such chemical modifications can be present throughout the polynucleotide or in an alternating pattern. Chemical modifications can be present at the 5' end or the 3' end or both, for example, 5-10 bases at the 5' end and / or 3' end contain one or more of modified backbone linkages, modified sugar moieties, modified phosphate moieties, modified nucleobases, or chemical conjugate moieties.

[0071] Such inhibitory polynucleotides can be delivered directly as a heterologous RNA or heterologous polynucleotide, e.g., a chemically modified polynucleotide that has been modified to increase its serum half-life and / or affinity. Alternatively, such inhibitory polynucleotides can be ectopically expressed from a DNA or vector that expresses such inhibitory polynucleotides. In another alternative, the endogenous expression of such inhibitory polynucleotides can be upregulated, e.g., by inserting an expression control sequence, such as a constitutive, inducible, strong, or tissue-specific promoter. Activating polynucleotide that increases SUV39H1 expression

[0072] Activating polynucleotides include reagents known in the art for inhibiting the expression of the lncRNA sequence or increasing its degradation, including RNA interference (RNAi), short hairpin RNA (shRNA), antisense oligonucleotide (ASO), or ribozyme, each of which contains a fragment complementary to the lncRNA sequence. Also contemplated are ZF or ZFN, TALE or TALEN, or CRISPR systems, such as CRISPR-Cas9 or CRISPR-Cas13, which contain a guide RNA, each guide RNA containing a fragment complementary to the lncRNA or the gene encoding or expressing the lncRNA. The length of the complementary region can be at least about 12-20, 12-18, or 15-18 bases.

[0073] Typically, after siRNA transfection or shRNA expression, RNAi triggers the degradation of the target RNA molecule through direct complementarity mediated by the RNA-induced silencing complex. An alternative to RNAi for degrading lncRNA is ASO( Figure 2B ). ASOs are single-stranded DNA oligomers of 15-20 nucleotides, which are typically chemically modified to enhance the potency of knockdown and reduce toxicity in vivo. In particular, 2'-MOE and LNA gapmer modifications have been shown to increase the affinity for the target RNA transcript and confer resistance to nucleases, resulting in a half-life of these modified ASOs in vivo of days to weeks. ASOs hybridize to the target NA transcript through complementarity and induce RNaseH-mediated degradation of the target transcript.

[0074] When provided with an sgRNA complementary to the target RNA, CRISPR-Cas13 can also effectively cleave the RNA target. Cas13 has been used to knockdown lncRNAs in mammalian cells. CRISPRi or zinc finger (ZF) or TALE proteins (where dCas9, TALE, and / or ZF are directly or indirectly linked to a repressor and / or inhibitor) can also inhibit RNA expression. Finally, the expression of lncRNAs can be eliminated by deleting or replacing all or part of the coding gene, such as by CRISPR-Cas9, ZF nuclease (ZFN), or TALE nuclease (TALEN) gene editing. See Liu and Lim, EMBO Reports (2018) 19: e46955. Expression of inhibitory polynucleotide or activating polynucleotide

[0075] The present disclosure also provides a nucleic acid that encodes or expresses (e.g., as a production template) AF196970.3 (SEQ ID NO: 1) or any one of SEQ ID NOs: 13-17 and 26-30, or a fragment or variant thereof that is capable of inhibiting SUV39H1 expression in a cell. In some embodiments, the nucleic acid is heterologous to the cell and is operably linked to an expression control sequence. In other embodiments, the nucleic acid is endogenous to the cell but is operably linked to a heterologous expression control sequence. Examples of such nucleic acids include the cDNA of ENSG00000232828 (SEQ ID NO: 5) or SEQ ID NO: 6, or the cDNA of SEQ ID NOs: 32-36 and 45-49, or a fragment or variant thereof. Other examples include nucleic acids encoding any one of exons 1-3 (SEQ ID NOs: 2-4), or a fragment or variant thereof. Other examples of such nucleic acids include any cDNA of SEQ ID NOs: 6-9, 32-36, and 45-49, or a fragment or variant thereof. Such nucleic acids can be part of a plasmid or vector or transposase system.

[0076] In some embodiments, the expression of an endogenous nucleic acid encoding or expressing AF196970.3 (SEQ ID NO: 1) or any one of the shRNAs described herein (SEQ ID NOs: 13-17 or 26-30), or a fragment or variant thereof that is capable of inhibiting SUV39H1 expression, is upregulated in a cell, e.g., by operably linking the endogenous nucleic acid to a heterologous expression control sequence (e.g., a promoter, enhancer, other regulatory sequence). In some embodiments, the expression of an endogenous gene or its allelic variant (e.g., ENSG00000232828 (SEQ ID NO: 5)) is upregulated in a cell, e.g., by operably linking the endogenous nucleic acid to a heterologous expression control sequence.

[0077] Methods and vectors for expressing polynucleotides (such as RNA) are well known in the art and are commercially available. Known vectors include viral vectors and pseudotyped viral vectors, such as retroviruses, lentiviruses, adenoviruses, adeno-associated viruses (AAV), alphaviruses, vaccinia viruses, poxviruses, EBV, herpes simplex virus, papillomaviruses, foamy viruses or Semliki Forest virus vectors, or transposase systems, such as the Sleeping Beauty transposase vector. Non-viral systems for delivering naked plasmids to cells include lipids, cationic lipid complexes, liposomes, nanoparticles, gold particles or polymer complexes, polylysine conjugates, synthetic polyamino polymers and artificial virus envelopes.

[0078] To express short non-coding RNAs, such as shRNA, polymerase III promoters are commonly used. Examples include the U6, H1 or 7SK promoters.

[0079] To express long RNAs (such as mRNA) or long non-coding RNAs, polymerase II promoters are commonly used. Examples include CMV, EF-1a, hPGK and RPBSA. The CAG promoter has been used for overexpressing lncRNA. Yin et al, Cell Stem Cell, May 7, 2015; 16(5): 504-16 (Yin et al Cell Stem Cell. 2015 May 7; 16(5): 504-16). Inducible promoters driven by signals from activated T cells include the nuclear factor of activated T cells (NFAT) promoter. Other promoters for T cell expression of RNA include the CIFT chimeric promoter (containing a cytomegalovirus (CMV) enhancer, the core interferon gamma (IFN-γ) promoter and a portion of the T lymphocyte virus long terminal repeat (TLTR)), the endogenous TRAC promoter or the TRBC promoter. Inducible, constitutive or tissue-specific promoters are considered.

[0080] Viral vectors have been used to overexpress lncRNAs, see Yang et al. (2013) Mol Cell 49: 1083 - 1096; Lu et al. (2018) Mol Ther Nucleic Acids 10: 387 - 397 (Yang et al. (2013) Mol Cell 49: 1083 - 1096; Lu et al. (2018) Mol Ther Nucleic Acids 10: 387 - 397). Additionally, lncRNAs have been expressed from lentiviral vectors and SB transposase systems. See, for example, Zhang et al., Overexpression of lncRNAs with endogenous lengths and functions using a lncRNA delivery system based on transposon. J Nanobiotechnol 19, 303 (2021) (Zhang et al., Overexpression of lncRNAs with endogenous lengths and functions using a lncRNA delivery system based on transposon. J Nanobiotechnol 19, 303 (2021)).

[0081] CRISPR - Cas9 - based activation systems have also been used to upregulate the endogenous expression of lncRNAs. Rankin et al., Overexpressing Long Noncoding RNAs Using Gene - activating CRISPR. J Vis Exp. 2019;(145):10.3791 / 59233 (Rankin et al., Overexpressing Long Noncoding RNAs Using Gene - activating CRISPR. J Vis Exp. 2019;(145):10.3791 / 59233).

[0082] In some embodiments, dsRNA (e.g., RNAi) is produced in a cell by a vector that contains an expression control sequence operably linked to a nucleotide sequence that expresses (as a template for one or both strands) the dsRNA. In other embodiments, promoters can be located at both ends of the template nucleotide sequence, where the promoters drive the expression of each individual DNA strand, thereby generating two complementary (or substantially complementary) RNAs that hybridize and form dsRNA. In other embodiments, dsRNA is produced in a cell by a vector that expresses shRNA, which is processed to form interfering dsRNA. Delivery of nucleic acid or polynucleotide

[0083] Techniques well recognized in the art for introducing exogenous nucleic acids (e.g., DNA and RNA) into host cells include calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, microinjection, DNA-loaded liposomes, lipofectamine-DNA complexes, sonication of cells, gene guns using high-velocity microparticles, biolistics, and virus-mediated transfection. Compositions containing nucleic acids may also contain transfection facilitators, which include surfactants, quinone analogs, vesicles (e.g., squalene and squalenes), hyaluronic acid, lipids, liposomes, phosphatidylcholine liposomes, calcium ions, viral proteins, polyanions, polycations, including poly-L-glutamic acid, or nanoparticles, gold particles, or other known reagents. Delivery vectors include liposomes, lipid-containing complexes, nanoparticles, gold particles, or polymer complexes.

[0084] Lipid materials have been used to fabricate ionizable cationic lipid-based lipid nanoparticles (LNPs), which exhibit a cationic charge at the reduced pH of late endosomes due to the tertiary amines in their structure to induce endosomal escape. These LNPs have been used, for example, to deliver RNA interference (RNAi) components as well as gene constructs or CRISPR-Cas systems. See, for example, Wilbie et al., Accounts of Chemical Research; 52(6):1555-1564, 2019. The use of biodegradable cationic LNPs was described by Wang et al., Proceedings of the National Academy of Sciences of the United States of America; 113(11):2868-2873, 2016. The use of ionizable lipids together with cholesterol, DSPC, and polyethylene glycolylated lipids to fabricate LNPs was described by Chang et al., Accounts of Chemical Research 52, 665-675, 2019 (Wilbie et al., Acc Chem Res.; 52(6):1555-1564, 2019. Wang et al., Proc Natl Acad Sci U S A.; 113(11):2868-2873, 2016describe use of biodegradable cationic LNPs.Chang et al., Acc.Chem.Res., 52, 665-675, 2019).

[0085] Polymeric-based particles can be used for gene construct delivery in a manner similar to lipids. Many materials have been used for nucleic acid delivery. For example, cationic polymers (such as polyethyleneimine (PEI)) can complex with nucleic acids and can induce endosomal uptake and release, similar to cationic lipids. The dendritic structure of polyamidoamine (PAMAM) can also be used for transfection. These particles consist of polymers from their branched core. Their surface shows cationic primary amines, which can complex with nucleic acids. Networks based on zinc to facilitate imidazole crosslinking have been used as a delivery method, relying on the low pH of late endosomes, which, after uptake, generate cationic charges due to the dissolution of zeolitic imidazolate frameworks (ZIFs), and subsequently these components are released into the cytosol. Colloidal gold nanoparticles have also been used. See Wilbie et al., supra. Production and chemical modification of inhibitory polynucleotide or activating polynucleotide

[0086] In vitro transcribed, chemically synthesized, or partially chemically synthesized RNA can be delivered. For example, RNA can be transcribed in vitro and chemically ligated to chemically synthesized RNA at the 5’ end and / or 3’ end. Direct injection or transfection of in vitro transcribed lncRNA has been performed to demonstrate the function of lncRNA. Ulitsky et al. (2011) Cell, 147(7):1537-1550.

[0087] Chemical modification of oligonucleotides (also known as polynucleotides) can improve their resistance to degradation, thus extending the half-life, and / or increasing the affinity for complementary polynucleotides. Modified oligonucleotides (e.g., RNA oligonucleotides) contain chemical modifications including one or more of modified backbone linkages, modified phosphate moieties, modified sugar moieties, modified nucleobases, or chemical conjugate moieties, or any combination thereof. Consider 1, 2, 3, 4, 5, 10, 15, 20, or more of the same type of modification, optionally combined with 1, 2, 3, 4, 5, 10, 15, 20, or more of another type of modification or modification pattern. Patterns include alternating modifications throughout the oligonucleotide, such as 2’-fluoro and 2’-methoxy, or terminal modifications, where, for example, 1, 2, 3, 4, 5, or more bases, sugars, or linkages at the 5’ end and / or 3’ end of the oligonucleotide are modified.

[0088] Examples of modified backbone linkages include phosphorothioates, phosphorothioate (PhTx) groups, or phosphonoacetates, phosphorothioacetates, methylphosphonates, boranophosphates, or dithiophosphates. Other internucleotide bridging modified phosphates can be used, such as methylthiophosphonates, morpholino phosphates, piperazino phosphates, and phosphoramidates. For example, each or every other internucleotide bridging phosphate residue can be modified as described.

[0089] Examples of modified sugar moieties include deoxyribose, or replacement of the 2′OH group with another group. While most modifications of sugar analogs occur at the 2’ position, other sites can also be modified, including the 4’ position. Example replacement groups include H, -OR, -R (where R can be alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), lower alkyl moieties (e.g., C1-C4, straight or branched, saturated or unsaturated alkyl such as methyl, ethyl, vinyl, propyl, 1-propenyl, 2-propenyl and isopropyl), halogen, -F, -Br, -Cl or -I, -SH, -SR (where R can be e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), -arabinosyl, F-arabinosyl, amino (where amino can be e.g., NH2; alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino, diheteroarylamino or amino acid); or cyano (-CN). Specific examples include 2’-fluorosugar, 2’-O-methyl sugar, 2’-O-methoxyethyl sugar or locked nucleic acid (LNA) nucleotides. Specific examples of modified 2’-sugars include 2’-F or 2’-O-methyl, adenosine (A), 2’-F or 2’-O-methyl, cytidine (C), 2’-F or 2’-O-methyl, uridine (U), 2’-F or 2’-O-methyl, thymidine (T), 2’-F or 2’-O-methyl, guanosine (G), 2’-O-methoxyethyl-5-methyluridine (Teo), 2’-O-methoxyethyladenosine (Aeo), 2’-O-methoxyethyl-5-methylcytidine (m5Ceo) and any combination thereof. For example, every other or each nucleotide can be modified as described.

[0090] Examples of modified nucleobases include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-hydroxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, methyl uracil-5-hydroxyacetate, uracil-5-hydroxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, and 2,6-diaminopurine.

[0091] Polynucleotides can also be stabilized by complexing with lipids or liposomes. In some embodiments, the liposome comprises 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In certain embodiments, the lipid particle comprises cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), PEG-cDMA or PEG-cDSA, and 1,2-dilinoleyloxy-3-(N,N-dimethyl)aminopropane (DLinDMA). Cell in which SUV39H1 expression is regulated

[0092] Cells according to the present disclosure exhibit regulation, preferably inhibition, of SUV39H1 expression. The cells are generally mammalian cells or cell lines, such as mouse, rat, pig, non-human primate, or preferably human. Such cells include cells derived from blood, bone marrow, lymph or lymphoid organs (especially the thymus), and are preferably cells of the immune system (i.e., immune cells), such as cells of innate or adaptive immunity, such as myeloid cells or lymphoid cells, including monocytes, macrophages, dendritic cells or lymphocytes, typically T cells and / or NK cells. The immune cells or their progenitors preferably also comprise one or more, or two or more, or three or more of the antigen-specific receptors (CAR and / or TCR) described herein, and optionally comprise one or more co-stimulatory receptors. The antigen-specific receptors according to the present disclosure include recombinantly modified T cell receptors (TCR) and their components, and functional non-TCR antigen-specific receptors, such as chimeric antigen receptors (CAR).

[0093] Cells according to the present disclosure can also be immune cell progenitors, such as lymphoid progenitors, and more preferably T cell progenitors. Examples of T cell progenitors include pluripotent stem cells (PSC), induced pluripotent stem cells (iPSC), hematopoietic stem cells (HSC), human embryonic stem cells (ESC), adipose stem cells (ADSC), multipotent progenitors (MPP), lymphoid-primed multipotent progenitors (LMPP), common lymphoid progenitors (CLP), lymphoid progenitors (LP), thymus-seeding progenitors (TSP) or early thymic progenitors (ETP). Hematopoietic stem cells and progenitors can be obtained from, for example, umbilical cord blood or peripheral blood, such as CD34+ cells derived from peripheral blood after mobilization treatment with granulocyte colony-stimulating factor (G-CSF). T cell progenitors generally express a set of consensus markers, including CD44, CD117, CD135 and / or Sca-1.

[0094] In some embodiments, the cells include one or more subsets of T cells or other cell types, such as the entire T cell population, CD4+ and / or CD8+ T cells and their subsets, such as subsets defined according to function, activation status, maturity, differentiation potential, expansion, recirculation, localization and / or persistence, antigen specificity, type of antigen-specific receptor, presence in a particular organ or compartment, marker or cytokine secretion profile and / or degree of differentiation. In some embodiments, the cells include myeloid-derived cells, such as dendritic cells, monocytes or macrophages.

[0095] Subtypes and subsets of T cells and / or CD4+ and / or CD8+ T cells include naive T (TN) cells, effector T cells (TEFF), memory T cells and their subtypes, such as stem cell memory T cells (TSCM), central memory T cells (TCM), effector memory T cells (TEM) or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, α / β T cells and δ / γ T cells. Specifically contemplated herein are TEFF cells having stem / memory properties and higher reconstitution capacity due to inhibition of SUV39H1, as well as TN cells, TSCM, TCM, TEM cells and combinations thereof.

[0096] In some embodiments, one or more T cell populations are enriched or depleted of cells that are positive or express high levels of one or more specific markers (such as surface markers), or negative or express relatively low levels of one or more markers. In certain cases, such markers are absent or expressed at relatively low levels in certain T cell populations (such as non-memory cells), but are present or expressed at relatively high levels in certain other T cell populations (such as memory cells). In one embodiment, cells (such as CD8+ cells or T cells, such as, CD3+ cells) are enriched (i.e., positively selected) for cells that are positive or express high surface levels of CD117, CD135, CD45RO, CCR7, CD28, CD27, CD44, CD127 and / or CD62L and / or depleted (e.g., negatively selected) for cells that are positive or express high surface levels of CD45RA. In some embodiments, cells are enriched or depleted of cells that are positive or express high surface levels of CD122, CD95, CD25, CD27 and / or IL7-Ra (CD127). In some instances, CD8+ T cells are enriched for cells that are positive for CD45RO (or negative for CD45RA) and positive for CD62LO. Subsets of CCR7+, CD45RO+, CD27+, CD62L+ cells constitute the central memory cell subset.

[0097] For example, according to the present disclosure, the cells can include a CD4+ T cell population and / or a CD8+ T cell subset, such as a subset enriched for central memory (TCM) cells. Alternatively, the cells can be other types of lymphocytes, including natural killer (NK) cells, mucosa-associated invariant T (MAIT) cells, innate lymphoid cells (ILC) and B cells.

[0098] The cells include primary cells directly isolated from a biological sample obtained from a subject and optionally cryopreserved. In some embodiments, the subject requires cell therapy (adoptive cell therapy) and / or will receive cell therapy. For a subject to be treated with cell therapy, the cells can be allogeneic and / or autologous. In autologous immune cell therapy, immune cells are collected from a patient, modified as described herein, and then returned to the patient. In allogeneic immune cell therapy, immune cells are collected from a healthy donor rather than the patient, modified as described herein, and then administered to the patient. Typically, these are HLA-matched to reduce the likelihood of host rejection. The immune cells can also include modifications that reduce immunogenicity, such as disrupting or removing HLA class I molecules, the HLA-A locus, and / or beta-2 microglobulin (B2M).

[0099] Off-the-shelf "ready-to-use" immune cells generally include modifications designed to reduce graft-versus-host disease, such as disruption or deletion of the endogenous TCR. Since a single gene encodes the c-chain (TRAC), as opposed to two genes encoding the beta-chain (TRBC), the TRAC locus is a common target for removing or disrupting endogenous TCR expression.

[0100] The sample includes a tissue sample from a tissue or organ, or a fluid sample, such as blood, plasma, serum, cerebrospinal fluid, or synovial fluid. The sample can be collected directly from the subject or obtained through one or more processing steps, such as separation, centrifugation, genetic engineering (e.g., transduction with a viral vector), washing, and / or incubation. A blood or blood-derived sample can be derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), white blood cells, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, myeloid-derived cells, and / or cells derived therefrom. Method for producing cells

[0101] The present disclosure provides methods for producing the disclosed cells with regulated SUV39H1 expression. For cells in which SUV39H1 expression is inhibited, such methods include introducing into such cells one or more, or two or more, or three or more of the inhibitory polynucleotides disclosed herein. More specifically, such methods include introducing into the cell a polynucleotide comprising the nucleobase sequence of AF196970.3 (SEQ ID NO: 1) or a fragment or variant thereof that is capable of inhibiting SUV39H1 expression in the cell, in an amount and under conditions effective to increase SUV39H1 expression, as described herein. Other examples include polynucleotides comprising the nucleobase sequence of any one of exons 1-3 (SEQ ID NOs: 2-4) or a fragment or variant thereof that is capable of inhibiting SUV39H1 expression in the cell. Such polynucleotides can be chemically modified to reduce degradation and / or increase affinity.

[0102] Such methods also include introducing into the cell a nucleic acid, plasmid, vector, or transposase system (preferably operably linked to an expression control sequence) encoding or expressing AF196970.3 (SEQ ID NO: 1) or an shRNA described herein (comprising any one of SEQ ID NOs: 13-17 or 26-30 described herein) or a fragment or variant thereof that is capable of inhibiting SUV39H1 expression in the cell, in an amount and under conditions effective to increase SUV39H1 expression. Examples of such nucleic acids include cDNA of ENSG00000232828 (SEQ ID NO: 5) or SEQ ID NOs: 6, 32-36, and 45-49 or fragments or variants thereof. Other examples include nucleic acids encoding any one of exons 1-3 (SEQ ID NOs: 2-4) or fragments or variants thereof. Other examples of such nucleic acids include any cDNA of SEQ ID NOs: 6-9 or fragments or variants thereof.

[0103] Such methods also include upregulating the endogenous expression of a nucleic acid encoding or expressing AF196970.3 (SEQ ID NO: 1) or an shRNA described herein (comprising any one of SEQ ID NOs: 13-17 and 26-30 described herein) or a fragment or variant thereof that is capable of inhibiting SUV39H1 expression in the cell, by inserting a heterologous expression control sequence (e.g., a promoter, enhancer, other regulatory sequence, constitutive, inducible, or tissue-specific) operably linked to the nucleic acid into the cell genome. In some embodiments, the expression of an endogenous gene or its allelic variant (e.g., ENSG00000232828 (SEQ ID NO: 5)) is upregulated in the cell.

[0104] Heterologous expression control sequences can be inserted into cells, for example, by homologous recombination with a donor template after cleavage with a known nuclease system (such as ZFN, TALEN, or a CRISPR system comprising a guide RNA (e.g., CRISPR-Cas9)).

[0105] For cells with increased SUV39H1 expression, such methods include introducing into such cells one or more, or two or more, or three or more of the activating polynucleotides described herein (such as RNAi, shRNA, ASO, ribozyme, or ZFN or TALEN or a CRISPR system comprising a guide RNA) in an amount and under conditions effective to increase SUV39H1 expression. Antigen-specific receptor

[0106] The cells with regulated SUV39H1 expression of the present disclosure include immune cells that express on their surface one or more, or two or more, or three or more antigen-specific receptors and optionally one or more co-stimulatory receptors. Antigen-specific receptors include recombinant or modified T cell receptors (TCRs) and their components, and / or chimeric antigen receptors (CARs). For example, at least two CARs, at least two TCRs, or at least one CAR and at least one TCR are contemplated. Antigen-specific receptors can bind the same or different antigens. In some embodiments, two or more antigen-specific receptors have different signaling domains. In some embodiments, the cell comprises an antigen-specific receptor with an activating signaling domain and an antigen-specific receptor with an inhibitory signaling domain. Generally, such antigen-specific receptors bind to the target antigen with a Kd binding affinity of about 10 - 6 M or lower, about 10 -7 M or lower, about 10 -8 M or lower, about 10 -9 M or lower, about 10 -10 M or lower or about 10 -11 M or lower (the lower the number, the higher the binding affinity) to the target antigen.

[0107] Thus, a cell can comprise one or more nucleic acids encoding one or more antigen - specific receptors, which are optionally operably linked to heterologous regulatory sequences. Typically, the nucleic acids are heterologous (i.e., not normally present in the engineered cell and / or the organism from which such cell is derived). In some embodiments, the nucleic acids are not naturally occurring and include chimeric combinations of nucleic acids encoding various domains from multiple different cell types. The nucleic acids and their regulatory sequences are typically heterologous. For example, a nucleic acid encoding an antigen - specific receptor can be heterologous to an immune cell and operably linked to an endogenous promoter of a T - cell receptor such that its expression is controlled by the endogenous promoter. In some embodiments, the nucleic acid encoding a CAR is operably linked to the endogenous TRAC promoter.

[0108] Immune cells (especially allogeneic immune cells) can be engineered to reduce graft - versus - host disease such that the cells comprise inactivated (e.g., disrupted or deleted) endogenous TCRs. Since a single gene encodes the α - chain (TRAC), as opposed to two genes encoding the β - chain, the TRAC locus is a typical target for reducing TCR receptor expression. Thus, a nucleic acid encoding an antigen - specific receptor (e.g., a CAR or a TCR) can be integrated at a position where the expression of the functional TCRα - chain is significantly reduced, preferably in the 5′ region of the first exon (SEQ ID NO: 3). See, for example, Jantz et al., WO 2017 / 062451; Sadelain et al., WO 2017 / 180989; Torikai et al., Blood, 119(2): 5697 - 705 (2012); Eyquem et al., Nature, Mar 2, 2017; 543(7643): 113 - 117 (Jantz et al., WO 2017 / 062451; Sadelain et al., WO 2017 / 180989; Torikai et al., Blood, 119(2): 5697 - 705 (2012); Eyquem et al., Nature. 2017 Mar 2; 543(7643): 113 - 117). The expression of the endogenous TCRα can be reduced by at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In such embodiments, the expression of the nucleic acid encoding the antigen - specific receptor is optionally controlled by the endogenous TCR - α promoter. Chimeric antigen receptor (CAR)

[0109] In some embodiments, the engineered antigen-specific receptors include chimeric antigen receptors (CARs), including activating or stimulatory CARs, co-stimulatory CARs (see WO2014 / 055668) and / or inhibitory CARs (iCARs, see Fedorov et al., Sci. Transl. Med. 5(215) (December 2013)).

[0110] A chimeric antigen receptor (CAR) (also known as a chimeric immune receptor, chimeric T cell receptor, artificial T cell receptor) is an engineered antigen-specific receptor that confers any specificity onto an immune effector cell (T cell). Typically, these receptors are used to confer the specificity of a monoclonal antibody onto a T cell and facilitate the transfer of its coding sequence via a retroviral vector.

[0111] A CAR generally comprises an extracellular antigen (or ligand) binding domain that is linked in some aspects via a linker and / or transmembrane domain to one or more intracellular signaling components. Such molecules generally mimic or approximate signals sent through the natural antigen receptor, signals sent through the binding of such a receptor to a co-stimulatory receptor, and / or signals sent through the co-stimulatory receptor alone.

[0112] A CAR can comprise (a) an extracellular antigen binding domain, (b) a transmembrane domain, (c) optionally a co-stimulatory domain, and (d) an intracellular signaling domain.

[0113] In some embodiments, a CAR is constructed to be specific for a particular antigen (or marker or ligand), such as an antigen expressed in a particular cell type targeted by adoptive cell therapy, such as a cancer marker. A CAR generally includes one or more antigen binding molecules in its extracellular portion, such as one or more antigen binding fragments, domains, or antibody portions, typically one or more antibody variable domains. For example, the extracellular antigen binding domain can comprise a light chain variable domain or a fragment thereof and / or a heavy chain variable domain or a fragment thereof, typically an scFv. In some embodiments, the CAR comprises a heavy chain variable domain or a fragment thereof that specifically binds an antigen.

[0114] The portions for binding an antigen include three classes: single-chain antibody fragments (scFv) derived from antibodies, Fab selected from a library, or a native ligand that binds its cognate receptor (for first-generation CARs). Successful examples of each of these classes are reported specifically in Sadelain M, Brentjens R, Riviere I., The basic principles of chimeric antigen receptor (CAR) design, Cancer discovery 2013; 3(4): 388-398 (Sadelain M, Brentjens R, Riviere I. The basic principles of chimeric antigen receptor (CAR) design. Cancer discovery. 2013; 3(4): 388-398) (see specifically Table 1) and are incorporated herein by reference.

[0115] Antibodies include chimeric antibodies, humanized antibodies, or human antibodies and can be further affinity matured and selected as described above. Chimeric or humanized scFvs derived from rodent immunoglobulins (e.g., mouse, rat) are commonly used because they are readily derived from well-characterized monoclonal antibodies. Humanized antibodies contain CDR regions derived from rodent sequences. Typically, rodent CDRs are grafted into a human framework and some human framework residues can be backmutated to the original rodent framework residues to maintain affinity, and / or one or several CDR residues can be mutated to increase affinity. Fully human antibodies have no murine sequences and are typically produced by phage display technology of human antibody libraries or immunization of transgenic mice (whose native immunoglobulin loci have been replaced by fragments of human immunoglobulin loci). Antibody variants having one or more amino acid substitutions, insertions, or deletions in their native amino acid sequences can be produced, wherein the antibody retains or substantially retains its specific binding function. Conservative substitutions of amino acids are well known and are as described above. Other variants having higher affinity for the antigen can also be produced.

[0116] In some embodiments, the modified TCR or CAR comprises an antibody fragment or antigen-binding fragment (e.g., scFv, or variable heavy chain (VH) region or variable light chain (VL) region or 1, 2, or 3 CDRs of such VH and / or VL) that specifically recognizes an intracellular antigen (e.g., a tumor-associated antigen) present on the cell surface as an MHC-peptide complex. Typically, a CAR containing an antibody or antigen-binding fragment that exhibits TCR-like specificity for a peptide-MHC complex can also be referred to as a TCR-like CAR.

[0117] In some embodiments, the transmembrane domain is derived from a natural or synthetic source. The transmembrane domain can be derived from the same receptor as the intracellular signaling domain or from a different receptor. The transmembrane region includes the transmembrane regions derived from (i.e., comprising at least their transmembrane regions) the α, β, or ζ chains of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS or GITR, or NKG2D, OX40, 2B4, DAP10, DAP12 or CD40. For T cells, CD8, CD28, CD3ε may be preferred. For NK cells, NKG2D, DAP10, DAP12 may be preferred. In some embodiments, the transmembrane domain is derived from CD28, CD8 or CD3ζ. In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

[0118] In some embodiments, there is a short oligopeptide or polypeptide linker, such as a linker having a length between 2 and 10 amino acids, and forms a connection between the transmembrane domain and the cytoplasmic signaling domain of the CAR.

[0119] CAR generally includes at least one or more intracellular signaling components. First-generation CARs typically have an intracellular domain from the CD3ζ chain, which is the main transmitter of signals from the endogenous TCR. Second-generation CARs generally also contain an intracellular signaling domain from various co-stimulatory protein receptors to the cytoplasmic tail region of the CAR to provide additional signals to the T cell. The co-stimulatory domain includes domains derived from human CD28, 4-1BB (CD137), ICOS, CD27, OX40 (CD134), DAP10, DAP12, 2B4, CD40, FCER1G or GITR (AITR). For T cells, CD28, CD27, 4-1BB (CD137), ICOS may be preferred. For NK cells, DAP10, DAP12, 2B4 may be preferred. Combinations of two co-stimulatory domains are contemplated, such as CD28 and 4-1BB, or CD28 and OX40. Third-generation CARs incorporate multiple signaling domains, such as CD3ζ-CD28-4-1BB or CD3ζ-CD28-OX40, to enhance potency.

[0120] T cell activation has been described in some aspects as being mediated by two classes of cytoplasmic signaling sequences: sequences that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences), and sequences that act in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences). In some aspects, a CAR includes one or both of such signaling components.

[0121] In some aspects, a CAR includes a primary cytoplasmic signaling sequence that modulates primary activation of the TCR complex in a stimulatory or inhibitory manner. A primary cytoplasmic signaling sequence that acts in a stimulatory manner can contain a signaling motif called an immunoreceptor tyrosine-based activation motif or ITAM. Examples of ITAMs that contain a primary cytoplasmic signaling sequence include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CDS, CD22, CD79a, CD79b, and CD66d. In some embodiments, the cytoplasmic signaling molecule in a CAR contains a cytoplasmic signaling domain, a portion thereof, or a sequence derived from CD3ζ. A CAR can also include the signaling domain and / or transmembrane portion of a co-stimulatory receptor (such as CD28, 4-1BB (CD137), ICOS, CD27, OX40 (CD134), DAP10, DAP12, 2B4, CD40, FCER1G, or GITR (AITR)). In some aspects, the same CAR includes an activation component and a co-stimulatory component; alternatively, the activation domain is provided by one CAR, and the co-stimulatory component is provided by another CAR that recognizes a different antigen.

[0122] The intracellular signaling domain can be from an intracellular component of the TCR complex, such as the TCR CD3 chains that mediate T cell activation and cytotoxicity, such as the CD3ζ chain. Alternative intracellular signaling domains include FcεRIγ. The intracellular signaling domain can comprise a modified CD3ζ polypeptide lacking one or two of its three immunoreceptor tyrosine-based activation motifs (ITAMs), where the ITAMs are ITAM1, ITAM2, and ITAM3 (numbered from the N-terminus to the C-terminus). The intracellular signaling region of CD3ζ is residues 22-164 of SEQ ID NO: 10. ITAM1 is around amino acid residues 61-89, ITAM2 is around amino acid residues 100-128, and ITAM3 is around residues 131-159. Thus, the modified CD3ζ polypeptide can inactivate any one of ITAM1, ITAM2, or ITAM3, such as by disruption or deletion. Alternatively, the modified CD3ζ polypeptide can inactivate any two ITAMs, such as ITAM2 and ITAM3, or ITAM1 and ITAM2. Preferably, ITAM3 is inactivated, such as by deletion. More preferably, ITAM2 and ITAM3 are inactivated, such as by deletion, leaving ITAM1. For example, a modified CD3ζ polypeptide retains only ITAM1 and the remaining CD3ζ domain is deleted (residues 90-164). As another example, ITAM1 is replaced with the amino acid sequence of ITAM3 and the remaining CD3ζ domain is deleted (residues 90-164). See, for example, Bridgeman et al., Clin. Exp. Immunol. 175(2):258-67 (2014); Zhao et al., J. Immunol. 183(9):5563-74 (2009); Maus et al., WO-2018 / 132506; Sadelain et al., WO-2019 / 133969, Feucht et al., Nat Med. 25(1):82-88 (2019) (Bridgeman et al., Clin. Exp. Immunol. 175(2):258-67 (2014); Zhao et al., J. Immunol. 183(9):5563-74 (2009); Maus et al., WO-2018 / 132506; Sadelain et al., WO-2019 / 133969, Feucht et al., Nat Med. 25(1):82-88 (2019)).

[0123] Thus, in some aspects, the antigen-binding molecule is linked to one or more cell signaling modules. In some embodiments, the cell signaling module includes a CD3 transmembrane domain, a CD3 intracellular signaling domain, and / or other CD transmembrane domains. The CAR can also include a portion of one or more additional molecules (such as Fc receptor gamma, CD8, CD4, CD25, or CD16).

[0124] In some embodiments, after the CAR is linked, the cytoplasmic domain or intracellular signaling domain of the CAR activates at least one normal effector function or response of the corresponding non-engineered immune cell (usually a T cell). For example, the CAR can induce functions of T cells, such as cytolytic activity or T helper activity, secretion of cytokines or other factors.

[0125] The CAR or other antigen-specific receptor can also be an inhibitory CAR (such as an iCAR) and includes intracellular components that suppress or inhibit a response (such as an immune response). Examples of such intracellular signaling components are those found on immune checkpoint molecules, including PD-1, CTLA4, LAG3, BTLA, OX2R, TIM-3, TIGIT, LAIR-1, PGE2 receptor, or EP2 / 4 adenosine receptor (including A2AR). In some aspects, the engineered cell includes an inhibitory CAR that includes a signaling domain of or derived from such an inhibitory molecule such that it serves to inhibit the cell response. For example, such CARs are used to reduce the likelihood of off-target effects when the antigen recognized by the activating receptor (such as a CAR) is also expressed or may also be expressed on the surface of normal cells. TCR

[0126] In some embodiments, the antigen - specific receptor includes a recombinantly modified T - cell receptor (TCR) and / or a TCR cloned from a naturally occurring T - cell. The nucleic acid encoding the TCR can be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of a naturally occurring TCR DNA sequence, followed by expression of the antibody variable region and then selection for specific binding to the antigen. In some embodiments, the TCR is obtained from T - cells isolated from a patient or a cultured T - cell hybridoma. In some embodiments, TCR clones of the target antigen have been generated in transgenic mice engineered with human immune system genes (e.g., the human leukocyte antigen system or HLA). See, e.g., tumor antigens (see, e.g., Parkhurst et al. (2009) Clin Cancer Res. 15:169 - 180 and Cohen et al. (2005) J Immunol. 175:5799 - 5808 (Parkhurst et al. (2009) Clin Cancer Res. 15:169 - 180 and Cohen et al. (2005) J Immunol. 175:5799 - 5808)). In some embodiments, phage display is used to isolate TCRs against the target antigen (see, e.g., Varela - Rohena et al. (2008) Nat Med. 14:1390 - 1395 and Li (2005) Nat Biotechnol. 23:349 - 354 (Varela - Rohena et al. (2008) Nat Med. 14:1390 - 1395 and Li (2005) Nat Biotechnol. 23:349 - 354)).

[0127] The "T - cell receptor" or "TCR" refers to a molecule containing variable alpha and beta chains (also referred to as TCRα and TCRβ, respectively) or variable gamma and delta chains (also referred to as TCRγ and TCRδ, respectively) and capable of specifically binding an antigenic peptide that binds to an MHC receptor. In some embodiments, the antigen - binding domain of the TCR has an affinity of about 1 x 10 -7 or lower, about 5 x 10 -8 or lower, about 1x 10 -8 or lower, about 5 x 10 -9 or lower, about 1 x 10 -9 or lower, about 5 x 10 -10 or lower, about 1 x 10 -10 or lower, about 5 x 10 -11 or lower, about 1 x 10 -11 or lower, about 5 x 10 -12 or lower or about 1 x 10 -12binds its target antigen with a KD affinity of or lower (where a lower number indicates a higher affinity). In some embodiments, the TCR is in the αβ form. Generally, TCRs that exist in the αβ and γδ forms are structurally similar, but the T cells that express them can have different anatomical locations or functions. The TCR can be present on the cell surface or in a soluble form. Generally, the TCR is located on the surface of a T cell (or T lymphocyte) and is typically responsible for recognizing an antigen bound to a major histocompatibility complex (MHC) molecule. In some embodiments, the TCR can also contain a constant domain, a transmembrane domain, and / or a short cytoplasmic tail region (see, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd Ed., Current Biology Publications, pp. 4:33, 1997). For example, in some aspects, each chain of the TCR can have an N-terminal immunoglobulin variable domain, an immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail region at the C-terminus. In some embodiments, the TCR is associated with invariant proteins of the CD3 complex that are involved in mediating signal transduction. Unless otherwise specified, the term "TCR" should be understood to encompass its functional TCR fragments. The term also encompasses full-length or modified TCRs, including TCRs in the αβ or γδ form. The term "TCR" also includes TCRs modified to contain the VH and / or VL of an antibody.

[0128] Thus, for the purposes of this disclosure, reference to a TCR includes any modified TCR or functional fragment thereof, such as the antigen-binding portion of a TCR that binds to a specific antigenic peptide bound in an MHC molecule, i.e., an MHC-peptide complex. The "antigen-binding portion" or "antigen-binding fragment" of a TCR is used interchangeably and refers to a molecule that contains a portion of the TCR domain but binds to the antigen (e.g., an MHC-peptide complex) to which the full-length TCR binds. In some cases, the antigen-binding portion contains the variable domains of the TCR, such as the variable α and variable β chains of the TCR, sufficient to form a binding site that binds to a specific MHC-peptide complex, e.g., typically each chain contains three complementarity-determining regions.

[0129] In some embodiments, the variable domains of the TCR chains associate to form loops, or immunoglobulin-like complementarity-determining regions (CDRs), which confer antigen recognition and define peptide specificity by forming the binding site of the TCR molecule. Typically, as with immunoglobulins, the CDRs are separated by framework regions (FRs) (see, e.g., Jores et al., Proc. Nat'l Acad. Sci. U.S.A. 87:9138, 1990; Chothia et al., EMBO J. 7:3745, 1988; see also Lefranc et al., Dev. Comp. Immunol. 27:55, 2003 (Jores et al., Proc. Nat'l Acad. Sci. U.S.A. 87:9138, 1990; Chothia et al., EMBO J. 7:3745, 1988; see also Lefranc et al., Dev. Comp. Immunol. 27:55, 2003)). In some embodiments, CDR3 is the major CDR responsible for recognition of processed antigen, although CDR1 of the α-chain has been shown to interact with the N-terminal portion of the antigenic peptide, and CDR1 of the β-chain interacts with the C-terminal portion of the peptide. CDR2 is thought to recognize MHC molecules. In some embodiments, the variable region of the β-chain may contain an additional hypervariable (HV4) region.

[0130] In some embodiments, the TCR chain comprises a constant domain. For example, like immunoglobulins, the extracellular portion of the TCR chain (e.g., the α chain, β chain) can comprise two immunoglobulin domains, a variable domain at the N-terminus (e.g., Vα or Vβ; typically amino acids 1 to 116 based on Kabat numbering Kabat et al., Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5th ed.) and a constant domain adjacent to the cell membrane (e.g., the α chain constant domain or Cα or TRAC, typically amino acids 117 to 259 based on Kabat, the β chain constant domain or Cβ or TRBC, typically amino acids 117 to 295). For example, in some cases, the extracellular portion of the TCR formed by two chains comprises two membrane-proximal constant domains and two membrane-distal variable domains containing CDRs. The constant domain of the TCR domain contains a short linker sequence in which cysteine residues form disulfide bonds to form a linkage between the two chains. In some embodiments, the TCR can have additional cysteine residues in each of the α and β chains such that the TCR contains two disulfide bonds in the constant domain.

[0131] In some embodiments, the TCR chain can comprise a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chain comprises a cytoplasmic tail region. In some cases, this structure allows the TCR to associate with other molecules (e.g., CD3). For example, a TCR containing a constant domain with a transmembrane region can anchor the protein in the cell membrane and associate with invariant subunits of the CD3 signaling apparatus or complex.

[0132] Typically, CD3 is a multi-protein complex that can have three different chains (gamma (γ), delta (δ), and epsilon (ε)) and the ζ chain. For example, in mammals, the complex can contain the CD3γ chain, the CD3δ chain, two CD3ε chains, and a homodimer of the CD3ζ chain. The CD3 γ chain is a highly related cell surface protein in the immunoglobulin superfamily and contains a single immunoglobulin domain. The transmembrane regions of the CD3γ, CD3δ, and CD3ε chains are negatively charged, a property that allows these chains to associate with the positively charged T cell receptor chains and play a role in propagating signals from the TCR into the cell. The intracellular tails of the CD3γ, CD3δ, and CD3ε chains each contain a single conserved motif called the immunoreceptor tyrosine-based activation motif or ITAM, while each CD3ζ chain has three ITAMs. Typically, ITAMs are involved in the signaling capacity of the TCR complex. The CD3γ, δ, ε, and ζ chains together form the so-called T cell receptor complex.

[0133] The modified TCRs of the present disclosure can comprise a heterologous antigen-binding domain and a native TCR constant domain (α or β) or a fragment thereof, wherein the modified TCR is capable of activating a CD3ζ polypeptide. Example modified TCRs, designated herein as HI-TCR or HIT-CAR, comprise (a) a first antigen-binding chain that comprises an antigen-binding fragment of an antibody heavy chain variable region (VH); and (b) a second antigen-binding chain that comprises an antigen-binding fragment of an antibody light chain variable region (VL); wherein the first and second antigen-binding chains each comprise a native or variant TRAC (constant region) or a fragment thereof, or a native or variant TRBC (constant region) or a fragment thereof. In some embodiments, at least one of the TRAC polypeptide and the TRBC polypeptide is endogenous, typically the TRAC polypeptide, and optionally one or both of the endogenous TRAC polypeptide and the TRBC polypeptide are inactivated.

[0134] In some designs, the HI-TCR comprises (a) a chimeric TCR α-chain comprising a VH or a fragment thereof fused to a native or variant TRAC or a fragment thereof, optionally wherein the amino acids of VH (or TRAC) are removed, and (b) a chimeric TCR β-chain comprising a VL or a fragment thereof fused to a native or variant TRBC or a fragment thereof, optionally wherein the amino acids of VL (or TRBC) are removed. In other designs, the HI-TCR comprises (a) a chimeric TCR α-chain comprising a VL or a fragment thereof fused to a native or variant TRAC or a fragment thereof, optionally wherein the amino acids of VL (or TRAC) are removed, and (b) a chimeric TCR β-chain comprising a VH or a fragment thereof fused to a native or variant TRBC or a fragment thereof, optionally wherein the amino acids of VH (or TRBC) are removed. In other designs, the HI-TCR comprises only a VH or a fragment thereof fused to a native or variant TRAC or a fragment thereof, or a VH or a fragment thereof fused to a native or variant TRBC or a fragment thereof, optionally wherein the amino acids of VH (or TRAC or TRBC) are removed. The HI-TCR (HIT-CAR) is described in International Patent Publication WO 2019 / 157454, which is incorporated herein by reference in its entirety. Other modified TCRs are described in International Patent Publication WO 2018 / 067993, which is incorporated herein by reference in its entirety, and Baeuerle et al., Synthetic TRuC receptors engage the complete T cell receptor to generate potent anti-tumor responses. Nat. Commun. 10, 2087 (2019) (Baeuerle, et al. Synthetic TRuC receptors engaging the complete T cell receptor for potent anti-tumor response. Nat Commun 10, 2087 (2019)). For example, any one or more, or two or more of the α, β, γ, or ε chains can be fused to an antibody variable region, such as VH and / or VL, such as scFv.

[0135] In some embodiments, a nucleic acid encoding a heterologous antigen-binding domain (e.g., VH or a variant or fragment thereof, or VL or a variant or fragment thereof) is inserted into the endogenous TRAC locus and / or the TRBC locus of an immune cell. Optionally, the nucleic acid encoding the chimeric TCR α (or β) chain is operably linked to an endogenous promoter of the T cell receptor such that its expression is controlled by the endogenous promoter. Insertion of the nucleic acid sequence can also inactivate or disrupt the endogenous expression of the TCR comprising the native TCR α-chain and / or the native TCR β-chain. Insertion of the nucleic acid sequence can reduce endogenous TCR expression by at least about 75%, 80%, 85%, 90%, or 95%.

[0136] When the density of antigen on the cell surface is low (less than about 10,000 molecules per cell, such as less than about 5,000, 4,000, 3,000, 2,000, 1,000, 500, 250, or 100 molecules per cell), immune cells comprising a recombinant TCR generally provide excellent activity. In some embodiments, the antigen is expressed at low density in a target cell, such as less than about 6,000 target antigen molecules per cell. In some embodiments, the antigen is expressed at a density of less than about 5,000 target antigen molecules per cell, less than about 4,000 molecules per cell, less than about 3,000 molecules per cell, less than about 2,000 molecules per cell, less than about 1,000 molecules per cell, or less than about 500 molecules per cell. In some embodiments, the antigen is expressed at a density of less than about 2,000 target antigen molecules per cell, such as less than about 1,800 molecules per cell, less than about 1,600 molecules per cell, less than about 1,400 molecules per cell, less than about 1,200 molecules per cell, less than about 1,000 molecules per cell, less than about 800 molecules per cell, less than about 600 molecules per cell, less than about 400 molecules per cell, less than about 200 molecules per cell, or less than about 100 molecules per cell. In some embodiments, the antigen is expressed at a density of less than about 1,000 target antigen molecules per cell, such as less than about 900 molecules per cell, less than about 800 molecules per cell, less than about 700 molecules per cell, less than about 600 molecules per cell, less than about 500 molecules per cell, less than about 400 molecules per cell, less than about 300 molecules per cell, less than about 200 molecules per cell, or less than about 100 molecules per cell. In some embodiments, the antigen is expressed at a density of about 5,000 to about 100 target antigen molecules per cell, such as about 5,000 to about 1,000 target antigen molecules per cell, about 4,000 to about 2,000 molecules per cell, about 3,000 to about 2,000 molecules per cell, about 4,000 to about 3,000 molecules per cell, about 3,000 to about 1,000 molecules per cell, about 2,000 to about 1,000 molecules per cell, about 1,000 to about 500 molecules per cell, about 500 to about 100 molecules per cell. In some embodiments, recombinant TCR T cell therapy targets antigens that are expressed at a lower density compared to their density in wild-type cells.

[0137] Other examples of antigen - specific receptors (including CARs and recombinant modified TCRs), and methods for engineering receptors and introducing them into cells, include those described in, for example, International Patent Application Publications WO - 2000 / 014257, WO - 2013 / 126726, WO - 2012 / 129514, WO - 2014 / 031687, WO - 2013 / 166321, WO - 2013 / 071154, WO - 2013 / 123061, U.S. Patent Application Publications US - 2002131960, US - 2013287748, US - 20130149337, U.S. Patents: 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European Patent Application EP2537416, and / or those described by Sadelain et al., Cancer Discovery, April 2013; 3(4):388 - 398; Davila et al. (2013) PLoS ONE 8(4):e61338; Turtle et al., Current Opinion in Immunology, October 2012; 24(5):633 - 39; Wu et al., Cancer, March 2012, 18(2):160 - 75 (Sadelain et al., Cancer Discov. 2013 April; 3(4):388 - 398; Davila et al., PLoS ONE 8(4):e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5):633 - 39; Wu et al., Cancer, 2012 March 18(2):160 - 75). In some aspects, the antigen - specific receptors include CARs as described in U.S. Patent 7,446,190, and those described in International Patent Application Publication WO - 2014 / 055668A1. Co-stimulatory receptor

[0138] The cells of the present disclosure having modified SUV39H1 expression may also comprise at least one or at least two exogenous co - stimulatory ligands. Co - stimulatory ligands include CD80, CD86, 4 - 1BBL, CD275, CD40L, OX40L, or any combination thereof. In some embodiments, the co - stimulatory ligand is CD80 or 4 - 1BBL.

[0139] In some embodiments, the cell comprises at least one or at least two costimulatory receptors. Such costimulatory receptors include chimeric receptors that comprise a costimulatory ligand fused to at least one or at least two costimulatory molecules. Costimulatory ligands include CD80, CD86, 4-1BBL, CD275, CD40L, OX40L, or any combination thereof. In some embodiments, the costimulatory ligand is CD80 or 4-1BBL. Exemplary costimulatory molecules are CD28, 4-1BB, 0X40, ICOS, DAP-10, CD27, CD40, NKG2D, CD2, or any combination thereof. In some embodiments, the chimeric receptor comprises a first costimulatory molecule 4-1BB and a second costimulatory molecule CD28.

[0140] In some embodiments, the cell comprises a costimulatory receptor that comprises an extracellular domain of CD80, a transmembrane domain of CD80, and an intracellular 4-1BB domain. Exemplary costimulatory ligands, molecules, and receptors (or fusion polypeptides) are described in International Patent Publication WO-2021 / 016174, which is incorporated herein by reference in its entirety.

[0141] The cells of the present disclosure having modified SUV39H1 expression may further comprise a T cell-specific engager, such as a BiTE, or a bispecific antibody that binds not only the desired antigen but also an activated T cell antigen, such as CD3ε. In some embodiments, the BiTe comprises an antigen-binding domain, such as an scFv, that is linked to a T cell recognition domain, such as the heavy chain variable domain and / or the light chain variable domain of an anti-CD3 antibody. Antigen

[0142] Antigens include antigens associated with a disease or disorder, including proliferative, neoplastic, and malignant diseases and disorders, more particularly cancer. Infectious diseases and autoimmune, inflammatory, or allergic diseases are also contemplated.

[0143] The cancer can be a solid cancer or a "liquid tumor", such as cancers affecting the blood, bone marrow, and lymphatic system, also known as hematopoietic and lymphoid tissue tumors, particularly including leukemia and lymphoma. Liquid tumors include, for example, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL) (including various lymphomas, such as mantle cell lymphoma, non-Hodgkin lymphoma (NHL), adenoma, squamous cell carcinoma, laryngeal cancer, gallbladder and bile duct cancer, retinal cancer, such as retinoblastoma).

[0144] Solid cancers specifically include cancers affecting one of the organs selected from the group consisting of: colon, rectum, skin, endometrium, lung (including non-small cell lung cancer), uterus, bone (such as osteosarcoma, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, giant cell tumor, ameloblastoma, and chordoma), liver, kidney, esophagus, stomach, bladder, pancreas, cervix, brain (such as meningioma, glioblastoma, low-grade astrocytoma, oligodendroglioma, pituitary tumor, schwannoma, and metastatic brain cancer), ovary, breast, head and neck, testis, prostate, and thyroid.

[0145] Cancers include cancers affecting the blood, bone marrow, and lymphatic system as described above. In some embodiments, the cancer is multiple myeloma or related to multiple myeloma. Antigens related to multiple myeloma include CD38, CD138, and / or CS-1. Other exemplary multiple myeloma antigens include CD56, TIM-3, CD33, CD123, and / or CD44.

[0146] The diseases also include infectious diseases or disorders, such as but not limited to viral, retroviral, bacterial, protozoal, or parasitic infections, HIV immunodeficiency, cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus, BK polyomavirus. Viral antigens include HIV, HCV, HBV antigens.

[0147] In some embodiments, the extracellular antigen-binding domain binds to any tumor neoantigen peptide disclosed in International Patent Publication WO 2021 / 043804, which is incorporated herein by reference in its entirety. For example, the antigen-binding domain binds to any peptide of SEQ ID NO: 1-117, or to a neoantigen peptide comprising at least 8, 9, 10, 11, or 12 amino acids, which is encoded by a portion of the open reading frame (ORF) of any fusion transcript sequence of SEQ ID NO: 118-17492 of WO 2021 / 043804.

[0148] The diseases also include autoimmune or inflammatory diseases or disorders, such as arthritis, such as rheumatoid arthritis (RA), type I diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, psoriasis, scleroderma, autoimmune thyroid disease, Grave's disease, Crohn's disease, multiple sclerosis, asthma, and / or diseases or disorders related to transplantation. In such cases, the regulatory T cells can be cells in which SUV39H1 is inhibited.

[0149] In some embodiments, the antigen is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen is selectively expressed or overexpressed on the cells of a disease or disorder (e.g., a tumor or pathogenic cell) compared to normal or non-target cells or tissues. In other embodiments, the antigen is expressed on normal cells and / or on engineered cells. In some such embodiments, the multi-targeting and / or gene disruption methods provided herein are used to increase specificity and / or potency.

[0150] In some embodiments, the antigen is a common tumor antigen. The term "common tumor antigen" refers to an immunogenic molecule, such as a protein, that is typically expressed at a higher level in tumor cells than in non-tumor cells and is also expressed in tumors of different origins. In some embodiments, the common tumor antigen is expressed in more than 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% or more of human cancers. In some embodiments, the common tumor antigen is expressed in at least three, at least four, at least five, at least six, at least seven, at least eight or more different types of tumors. In some cases, the common tumor antigen may be expressed in non-tumor cells (e.g., normal cells), but at a lower level than its expression level in tumor cells. In some cases, the common tumor antigen is not expressed at all in non-tumor cells, e.g., not expressed in normal cells. Exemplary common tumor antigens include, for example, human telomerase reverse transcriptase (hTERT), survivin, murine double minute 2 homolog (MDM2), cytochrome P450 1B1 (CYP1B), HER2 / neu, p95HER2, Wilms tumor gene 1 (WT1), livin, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), mucin 16 (MUC16), MUC1, prostate-specific membrane antigen (PSMA), p53 or cyclin (D1). Peptide epitopes of tumor antigens (including common tumor antigens) are known in the art and in some aspects can be used to generate MHC-restricted antigen-specific receptors, such as TCRs or TCR-like CARs (see, for example, published PCT applications WO-2011 / 009173 or WO-2012 / 135854 and published US application US-20140065708).

[0151] In some embodiments, the cancer is an overexpression of HER2 or p95HER2 or is associated with an overexpression of HER2 or p95HER2. p95HER2 is a constitutively active C-terminal fragment of HER2 that is generated by alternative initiation of translation at methionine 611 of the transcript encoding the full-length HER2 receptor. The amino acid sequence of p95HER2 is shown in SEQ ID NO: 11, and the amino acid sequence of the extracellular domain is shown in SEQ ID NO: 12.

[0152] It has been reported that HER2 or p95HER2 is overexpressed in breast cancer, gastric cancer, gastroesophageal cancer, esophageal cancer, ovarian cancer, endometrial cancer, cervical cancer, colon cancer, bladder cancer, lung cancer, and head and neck cancer. Cancer patients expressing the p95HER2 fragment are more likely to develop metastases and have a worse prognosis than those mainly expressing the full form of HER2. Saez et al., Clinical Cancer Research 12:424-431 (2006).

[0153] Other antigens include orphan tyrosine kinase receptor ROR1, tEGFR, Her2, p95HER2, LI-CAM, CD19, CD20, CD22, mesothelin, CEA, claudin 18.2, hepatitis B surface antigen, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, CD70, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, 3 or 4, FBP, FcRH5 fetal acetylcholine e receptor, GD2, GD3, HMW-MAA, IL-22R-c, IL-13R-α2, kdr, K light chain, Lewis Y, L1 cell adhesion molecule, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D ligand, NY-ESO-1, MART-1, gplOO, oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate specific antigen, PSMA, Her2 / neu, p95HER2, estrogen receptor, progesterone receptor, ephrinB2, CD 123, CS-1, c-Met, GD-2 and MAGE A3, CE7, Wilms tumor 1 (WT-1), cyclins such as cyclin A1 (CCNA1), and / or molecules expressed by HIV, HCV, HBV, or other pathogens.

[0154] In some embodiments, the recombinant nucleic acids encoding the antigen receptors are transferred into T cells by electroporation (see, e.g., Chicaybam et al., (2013) PLoS ONE 8(3): e60298 and VanTedeloo et al., (2000) Gene Therapy 7(16): 1431-1437). In some embodiments, recombinant nucleic acids are transferred into T cells via transposition (see, e.g., Manuri et al. (2010) Hum Gene Ther 21(4): 427-437; Sharma et al. (2013) Mol Ther Nucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506: 115-126). Other methods for introducing and expressing genetic material in immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y.), protoplast fusion, cationic lipid-mediated transfection; tungsten particle-assisted microprojectile bombardment (Johnston, Nature 346: 776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol. 7: 2031-2034 (1987)).

[0155] Particularly useful vectors for generating transgenic vectorized target constructs for homologous recombination-mediated targeting include, but are not limited to, recombinant adeno-associated virus (rAAV), recombinant non-integrating lentivirus (rNILV), recombinant non-integrating Y-retrovirus (rNIgRV), single-stranded DNA (linear or circular), and the like. Such vectors can be used to introduce transgenes into the immune cells of the present invention by preparing target constructs (see, for example, Miller, Hum., Gene Ther. 1(1):5-14 (1990); Friedman, Science 244:1275-1281 (1989); Eglitis et al., BioTechniques 6:608-614 (1988); Tolstoshev et al., Current Opin. Biotechno1. 1:55-61 (1990); Sharp, Lancet 337:1277-1278 (1991); Cornetta et al., Prog. Nucleic Acid Res. Mol. Biol. 36:311-322 (1989); Anderson, Science 226:401-409 (1984); Moen, Blood Cells 17:407-416 (1991); Miller et al., BioTechniques 7:980-990 (1989); Le Gal La Salle et al., Science 259:988-990 (1993); and Johnson, Chest 107:77S-83S (1995); Rosenberg et al., N. Engl. J. Med. 323:370 (1990); Anderson et al., U.S. Patent 5,399,346; Scholler et al., Sci. Transl. Med. 4132-153 (2012); Parente-Pereira et al., J. Biol. Methods 1(2):e7 (1-9) (2014); Lamers et al., Blood 117(1):72-82 (2011); Reviere et al., Proc. Natl. Acad. Sci. U.S.A. 92:6733-6737 (1995); Wang et al., Gene Ther. 15:1454-1459 (2008) (Miller, Hum. Gene Ther. 1(1):5-14 (1990); Friedman, Science 244:1275-1281 (1989); Eglitis et al., BioTechniques 6:608-614 (1988); Tolstoshev et al., Current Opin. Biotechno1. 1:55-61 (1990); Sharp, Lancet 337:1277-1278 (1991); Cornetta et al., Prog. Nucleic Acid Res. Mol. Biol. 36: 311 - 322 (1989); Anderson, Science 226: 401 - 409 (1984); Moen, Blood Cells 17: 407 - 416 (1991); Miller et al., Biotechnology 7: 980 - 990 (1989); Le Gal La Salle et al., Science 259: 988 - 990 (1993); and Johnson, Chest 107: 77S - 83S (1995); Rosenberg et al., N. Engl. J. Med. 323: 370 (1990); Anderson et al., U.S. Pat. No. 5,399,346; Scholler et al., Sci. Transl. Med. 4: 132 - 153 (2012; Parente - Pereira et al., J. Bio1. Methods l(2): e7(1 - 9)(2014); Lamers et al., Blood 117(1): 72 - 82 (2011); Reviere et al., Proc. Natl. Acad. Sci. USA 92: 6733 - 6737 (1995); Wang et al., Gene Therapy 15: 1454 - 1459 (2008))).

[0156] In some embodiments, the exogenous nucleic acid or targeting construct comprises a 5' homology arm and a 3' homology arm to facilitate recombination of a nucleic acid sequence into the cellular genome at a nuclease cleavage site.

[0157] In some embodiments, single-stranded DNA templates can be used to introduce exogenous nucleic acids into cells. The single-stranded DNA can contain the exogenous nucleic acid and, in preferred embodiments, can contain 5' and 3' homology arms to facilitate insertion of the nucleic acid sequence into the nuclease cleavage site by homologous recombination. The single-stranded DNA can also contain a 5' AAV inverted terminal repeat (ITR) sequence 5' upstream of the 5' homology arm and a 3' AAV ITR sequence 3' downstream of the 3' homology arm. In other specific embodiments, the targeting construct contains, in 5' to 3' order: a first viral sequence, a left homology arm, a nucleic acid sequence encoding elements that produce a polycistronic expression cassette (e.g., various viral and non-viral internal ribosome entry sites (IRESs, e.g., FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF-kB IRES, RUNX1 IRES, p53 IRES, hepatitis A IRES, hepatitis C IRES, pestivirus IRES, foot-and-mouth disease virus IRES, picornavirus IRES, poliovirus IRES, and encephalomyocarditis virus IRES) and a cleavable linker (e.g., 2A peptides, e.g., P2A, T2A, E2A, and F2A peptides, preferably a cleavable linker), a transgene, a polyadenylation sequence, a right homology arm, and a second viral sequence. In preferred embodiments, the targeting construct contains, in 5' to 3' order: a first viral sequence, a left homology arm, a nucleic acid sequence encoding a self-cleaving linker (e.g., porcine teschovirus 2A), a nucleic acid sequence encoding a CAR or a modified TCR (e.g., Hi-CTR), a polyadenylation sequence, a right homology arm, and a second viral sequence. Another suitable targeting construct can contain sequences from an integrase-deficient lentivirus (see, e.g., Wanisch et al., Mol. Ther. 17(8):1316-1332 (2009)).

[0158] In some embodiments, the viral nucleic acid sequence contains sequences from an integrase-deficient lentivirus. It should be understood that any suitable targeting construct compatible with the homologous recombination system employed can be utilized. The AAV nucleic acid sequences that function as part of the targeting construct can be packaged in several native or recombinant AAV capsids or particles. In one specific embodiment, the AAV particle is AAV6. In one specific embodiment, AAV6 viral particles are used to deliver an AAV2-based targeting construct to the target cell. In one specific embodiment, the AAV sequence is an AAV2, AAV5, or AAV6 sequence.

[0159] In some embodiments, the gene encoding the exogenous nucleic acid sequence of the present invention can be introduced into cells by transfection with a linearized DNA template. In some instances, the plasmid DNA encoding the exogenous nucleic acid sequence can comprise nuclease cleavage sites (e.g., class II, type II, type V, or type VI Cas nucleases) flanking the left homologous arm, such that the circular plasmid DNA is linearized and allows precise in-frame integration of the exogenous DNA without the backbone vector sequence (see, e.g., Hisano Y, Sakuma T, Nakade S, et al. Precise in-frame integration of exogenous DNA mediated by CRISPR / Cas9 system in zebrafish. Sci Rep. 2015;5:8841).

[0160] In some embodiments, the vector comprises an endogenous promoter, such as the TCR promoter. Such a vector can provide expression in a manner similar to that provided by an endogenous promoter (e.g., the TCR promoter). Such a vector can be useful, for example, if the integration site does not express the transgene efficiently, or if disruption of the endogenous gene controlled by the endogenous promoter is detrimental to the T cell or results in reduced efficacy in T cell therapy. In a preferred embodiment, such a vector can be useful, for example, if the integration site does not express the nucleic acid sequence encoding a CAR or a modified TCR efficiently. The promoter can be an inducible promoter or a constitutive promoter. Expression of the nucleic acid sequence under the control of an endogenous or vector-related promoter occurs under conditions suitable for cell expression of the nucleic acid, such as growth conditions, or in the presence of an inducer for an inducible promoter, etc. Those skilled in the art will understand such conditions.

[0161] The targeting construct can optionally be designed to include elements that generate a polycistronic expression cassette (including but not limited to various viral and non-viral internal ribosome entry sites (IRES, e.g., FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF-kB IRES, RUNX1 IRES, p53 IRES, hepatitis A IRES, hepatitis C IRES, pestivirus IRES, foot-and-mouth disease virus IRES, picornavirus IRES, poliovirus IRES, and encephalomyocarditis virus IRES) and a cleavable linker (e.g., 2A peptides, e.g., P2A, T2A, E2A, and F2A peptides) directly upstream of the nucleic acid sequence encoding the transgene). In a preferred embodiment, the targeting construct can optionally be designed to include a cleavable linker (e.g., P2A, T2A, etc.) sequence directly upstream of the nucleic acid sequence encoding a therapeutic protein (e.g., an engineered antigen receptor). P2A and T2A are self-cleaving peptide sequences that can be used for bicistronic or polycistronic expression of protein sequences (see Szymczak et al., Expert Opin. Biol. Ther. 5(5):627-638 (2005)).

[0162] A very suitable AAV construct for expressing HIT in immune response cells according to the present application is described, for example, in Mansilla-Soto, J., Eyquem, J., Haubner, S. et al., HLA-independent T cell receptors for targeting tumors with low antigen density, Nat. Med. 28, 345-352 (2022) (Mansilla-Soto, J., Eyquem, J., Haubner, S. et al. HLA-independent T cell receptors for targeting tumors with low antigen density. Nat Med 28, 345-352 (2022)), and has been used for the results included herein, particularly for in vivo experiments.

[0163] Typical well-suited constructs generally include a TRBC or TRAC sequence (which can be a native or modified TRBC or TRAC sequence, including murine sequences as described herein), a cleavable linker sequence (as defined above, but for example a 2A sequence), a TRAC or TRBC sequence (which can be a native or modified TRBC or TRAC sequence, including murine sequences as described herein). The TRBC and / or TRAC sequences are generally fused (preferably at the 5') to a sequence encoding the above antibody fragment (such as VH, VH, scFv, single domain antibody, VHH, etc.). In some embodiments, a costimulatory (co-stimulatory ligand) sequence is included in the construct such that in a preferred embodiment, the construct further includes a cleavable linker sequence (e.g., a 2A sequence) and a costimulatory (co-stimulatory ligand) sequence. Generally, the TRAC or TRB at the 3' end of the construct is fused to a cleavable linker, which is also fused to a costimulatory (co-stimulatory ligand and / or co-stimulatory receptor CCR) sequence (see Figure 11, and Figures 29 - 30). The costimulatory (co-stimulatory ligand and / or co-stimulatory receptor CCR) sequence can be any of those described herein and can in particular be the CD80 sequence or the CD80_4-1BB sequence as described herein (see for example SEQ ID NO: 32 - 33 and 52 - 53).

[0164] If desired, the targeting construct can optionally be designed to include a reporter gene, e.g., a reporter protein that identifies transduced cells. Exemplary reporter proteins include, but are not limited to, fluorescent proteins such as mCherry, green fluorescent protein (GFP), blue fluorescent proteins such as EBFP, EBFP2, Azurite and mKalamal, cyan fluorescent proteins such as ECFP, Cerulean and CyPet, and yellow fluorescent proteins such as YFP, Citrine, Venus and YPet. Generally, the targeting construct contains a polyadenylation (poly A) sequence 3' of the transgene. In a preferred embodiment, the targeting construct contains a polyadenylation (poly A) sequence at the 3' end of the nucleic acid sequence encoding a CAR and / or a modified TCR (such as a Hi-TCR). Therapeutic use

[0165] The cells, modified oligonucleotides, nucleic acids or vectors of the present disclosure can be used in adoptive cell therapy (particularly adoptive T cell therapy or adoptive NK cell therapy). In some embodiments, the use is for treating cancer in a subject in need thereof, but the use also includes treating infectious diseases and autoimmune, inflammatory or allergic diseases. In some embodiments, the subject has cancer or is at risk of developing cancer. The modified oligonucleotides, nucleic acids or vectors of the present disclosure are optionally within the delivery vectors or compositions disclosed herein, including liposomes, lipid-containing complexes, nanoparticles, gold particles or polymer complexes. Such compositions can also contain stabilizers or transfection promoters, such as surfactants, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, lecithin liposomes, calcium ions, viral proteins, polyanions, polycations, including poly-L-glutamic acid, or nanoparticles, gold particles or other known reagents.

[0166] In such methods, one or more of the cells, modified oligonucleotides, nucleic acids or vectors described herein are administered to a subject in need thereof in an amount effective to treat the disease or disorder. For example, cells expressing one or more antigen-specific receptors are administered in a dose effective to treat a disease or disorder associated with the antigen. Consider treating any of the diseases listed in the "Antigen" section above.

[0167] In some embodiments, immune cells expressing an antigen receptor described herein (e.g., a Hi T cell antigen receptor) can be used to treat patients with a median of less than about 6,000 target antigen molecules per cell. In some embodiments, the antigen is expressed at a density (typically median) of less than about 5,000 target antigen molecules per cell, less than about 4,000 molecules, less than about 3,000 molecules, less than about 2,000 molecules, less than about 1,000 molecules, or less than about 500 molecules. In some embodiments, the antigen is expressed at a density (typically median) of less than about 2,000 target antigen molecules per cell, e.g., less than about 1,800 molecules, less than about 1,600 molecules, less than about 1,400 molecules, less than about 1,200 molecules, less than about 1,000 molecules, less than about 800 molecules, less than about 600 molecules, less than about 400 molecules, less than about 200 molecules, or less than about 100 molecules. In some embodiments, the antigen is expressed at a density of less than about 1,000 target antigen molecules per cell, e.g., less than about 900 molecules, less than about 800 molecules, less than about 700 molecules, less than about 600 molecules, less than about 500 molecules, less than about 400 molecules, less than about 300 molecules, less than about 200 molecules, or less than about 100 molecules. In some embodiments, the antigen is expressed at a density of from about 5,000 to about 100 target antigen molecules per cell, e.g., from about 5,000 to about 1,000 target antigen molecules per cell, from about 4,000 to about 2,000 molecules, from about 3,000 to about 2,000 molecules, from about 4,000 to about 3,000 molecules, from about 3,000 to about 1,000 molecules, from about 2,000 to about 1,000 molecules, from about 1,000 to about 500 molecules, from about 500 to about 100 molecules.The target antigen density per cell can be quantified as described in Jasper, G.A., Arun, I., Venzon, D., Kreitman, R.J., Wayne, A.S., Yuan, C.M., Marti, G.E. & Stetler-Stevenson, M. (2011). Variables affecting the quantitation of CD22 in neoplastic B cells. Cytometry. Part B, Clinical cytometry, 80(2), 83-90.

[0168] Cells can be administered at a certain dose. For example, immune cells in which SUV39H1 is inhibited (e.g., T cells or NK cells) can be administered to an adult at a dose of less than about 10 8 cells, less than about 5 x 10 7 cells, less than 10 7 cells, less than about 5 x 10 6 cells, less than 10 6 cells, less than about 5 x 10 5 cells or less than about 10 5 cells. The dose for pediatric patients can be less than about 1 / 100. In alternative embodiments, any of the immune cells described herein (e.g., T cells) can be administered to a patient at a dose of from about 10 5 cells to about 10 9 cells, or from about 10 5 cells to about 10 8 cells, or from about 10 5 cells to about 10 7 cells, or from about 10 6 cells to about 10 8 cells.

[0169] The subject (i.e., the patient) is a mammal, typically a primate, such as a human. In some embodiments, the primate is a monkey or an ape. The subject can be male or female and can be of any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some embodiments, the subject is a non - primate mammal, such as a rodent. In some instances, the patient or subject is a validated animal model for a disease, adoptive cell therapy, and / or evaluation of toxic outcomes (e.g., cytokine release syndrome (CRS)). In some embodiments, the subject has cancer, is at risk of developing cancer, or is in cancer remission.

[0170] In some embodiments, the cells or compositions are administered to a subject, such as a subject having cancer or any of the aforementioned diseases or at risk of developing cancer or any of the aforementioned diseases. In some aspects, the method thereby treats, e.g., ameliorates one or more symptoms of a disease or disorder (e.g., in the case of cancer) by reducing the tumor burden in a cancer that expresses the antigen recognized by the engineered cells.

[0171] Methods of administering cells for adoptive cell therapy are known and can be used in combination with the provided methods and compositions. For example, methods of adoptive T - cell therapy are described in, e.g., U.S. Patent Application Publication 2003 / 0170238 to Gruenberg et al., U.S. Patent 4,690,915 to Rosenberg, and Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577 - 85. See, e.g., Themeli et al. (2013) Nat Biotechnol. 31(10):928 - 933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1):84 - 9; Davila et al. (2013) PLoS ONE 8(4):e61338 (Themeli et al. (2013) Nat Biotechnol. 31(10):928 - 933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1):84 - 9; Davila et al. (2013) PLoS ONE 8(4):e61338).

[0172] Administering at least one cell according to the present disclosure to a subject in need thereof can be combined with one or more additional therapeutic agents or in combination with another therapeutic intervention, and can be carried out simultaneously or sequentially in any order. In some cases, the cells and another therapy are co-administered close enough in time such that the cell population enhances the effect of one or more additional therapeutic agents, and vice versa. In some embodiments, the cell population is administered before one or more additional therapeutic agents. In some embodiments, the cell population is administered after one or more additional therapeutic agents.

[0173] Regarding cancer treatment, combined cancer treatment can include, but is not limited to, cancer chemotherapeutic agents, cytotoxic agents, hormones, anti-angiogenic agents, radiolabeled compounds, immunotherapy, surgery, cryotherapy, and / or radiotherapy.

[0174] Conventional cancer chemotherapeutic agents include alkylating agents, antimetabolites, anthracyclines, topoisomerase inhibitors, microtubule inhibitors, and B-raf enzyme inhibitors.

[0175] Alkylating agents include nitrogen mustards (such as dichloromethyldiethylamine, cyclophosphamide, ifosfamide, melphalan, and chlorambucil), ethylamine and methylamine derivatives (such as altretamine, thiotepa), alkyl sulfonic acids (such as busulfan), nitrosoureas (such as carmustine, lomustine, estramustine), triazenes (such as dacarbazine, procarbazine, temozolomide), and platinum-containing antitumor drugs (such as cisplatin, carboplatin, oxaliplatin).

[0176] Antimetabolites include 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine cytarabine fluorouridine, fludarabine, gemcitabine hydroxyurea, methotrexate, pemetrexed

[0177] Anthracyclines include daunorubicin, doxorubicin epirubicin, idarubicin. Other antitumor antibiotics include actinomycin-D, bleomycin, mitomycin-C, mitoxantrone.

[0178] Topoisomerase inhibitors include topotecan, irinotecan (CPT-11), etoposide (VP-16), teniposide, or mitoxantrone.

[0179] Microtubule inhibitors include estramustine, ixabepilone, taxanes (such as paclitaxel, docetaxel, and cabazitaxel), and vinca alkaloids (such as vinblastine, vincristine, vinorelbine, vindesine, and vinflunine).

[0180] B-raf enzyme inhibitors include vemurafenib (Zelboraf), dabrafenib (Tafinlar), and encorafenib (Braftovi).

[0181] Immunotherapies include, but are not limited to, immune checkpoint modulators (i.e., inhibitors and / or agonists), cytokines, immunomodulatory monoclonal antibodies, and cancer vaccines.

[0182] Preferably, the administration of cells in the adoptive T cell therapy according to the present disclosure is combined with the administration of an immune checkpoint modulator. Examples include inhibitors of PD-1, CTLA4, LAG 3, BTLA, OX2R, TIM-3, TIGIT, LAIR-1, PGE2 receptor, and / or EP2 / 4 adenosine receptor (including A2AR) (e.g., antibodies that specifically bind and inhibit their activity). Preferably, the immune checkpoint modulator includes anti-PD-1 and / or anti-PDL-1 inhibitors (e.g., anti-PD-1 and / or anti-PDL-1 antibodies).

[0183] The present disclosure also relates to the use of a composition comprising the cells described herein in the manufacture of a medicament for treating cancer, infectious disease or disorder, autoimmune disease or disorder, or inflammatory disease or disorder in a subject. Examples Example 1: Expression of lncRNA AF196970.3 in human tissues

[0184] The expression of lncRNA AF196970.3 was examined in various human tissues. AF196970.3 was expressed in many different healthy tissues, with relatively high expression in the cervix, brain, ovary, and uterus ( Figure 3A ), somewhat similar to SUV39H1 expression ( Figure 3B ). It was also detected in many different types of cells, with the highest levels in endothelial cells, fibroblasts, and myocytes (Figure 3B ), is partially similar to SUV39H1( Figure 3D ). Only one transcript of AF196970.3 (ENST00000416061.1) was found to be expressed in the GTEx project( Figure 3C ).

[0185] The expression of lncRNA AF196970.3 was examined in human T cells, especially in the context of cancer. AF196970.3 was found to be expressed in tumor-infiltrating lymphocytes in three different projects: glioma (Wang et al., 2020)( Figure 4A ), head and neck squamous cell carcinoma (Cillo et al., 2020)( Figure 4B ), and hepatocellular carcinoma (Zhang et al., 2019)( Figure 4C ). The expression level of AF196970.3 was correlated with the level of SUV39H1 and was higher in proliferating T cells( Figure 4B , Figure 4C ).

[0186] Primers were designed to measure the expression of AF196970.3 in human T cells freshly isolated from PBMCs and T cells from tumors (both activated or not activated in vitro) by RT-PCR. Expression patterns were detected. Example 2: Overexpression of lncRNA AF196970.3 in HEK293 cells

[0187] Figure 5 a and Figure 5 b respectively show the Piggy Bac backbone with the GFP-puromycin reporter gene, including the lncRNA SUV39H1 exon sequence with the CMV promoter or the hPKG promoter. lncRNA AF196970.3 was overexpressed in HEK293 FT cells to evaluate its effect on the expression and activity of SUV39H1.

[0188] Briefly, HEK293 cells were seeded with 5.10 5 cells on day 0. On day 1, the cells were transfected with the Piggy Bac (PB) construct and the Super PiggyBac transposase expression vector (using Purefection transfection reagent). On day 4, the medium was changed to remove the remaining reagents. Starting from day 7, transfected cells were selected with 1 μg / mL puromycin. On day 23, the cells were harvested and analyzed for the expression of GFP and SUV39H1. Figure 6 b shows the GFP expression levels determined by flow cytometry, and the comparison of untransfected cells with cells transfected with the empty Piggy Bac construct or one of the above lncRNA SUV39H1 Piggy Bac plasmids.Figure 6 d shows the quantification of SUV SUV39H1 protein level relative to actin. The results indicate that the SUV39H1 protein levels of both lncRNA constructs were decreased at day 23 post transfection compared with the PB empty vector. Example 3: Effect of lncRNA AF196970.3 overexpression on human T cells

[0189] AF196970.3 was cloned into the lncRNA expression vector, including PB-CAG-BGHpA (Addgene #92161) (Yin et al., Cell Stem Cell, 2015). The alternative vector system includes the ELECTS transposon system (see Zhang et al., Overexpression of lncRNAs with endogenous lengths and functions using a lncRNA delivery system based on transposon. J Nanobiotechnol. 19, 303 (2021))).

[0190] Figure 6 d describes the experimental procedures for overexpression of lncRNA Suv39h1 (AF196970.3) in CD8+ T cells. Briefly, T cell activation was performed on day 0. IL-2 was added on day 2. On day 3, cells were nucleofected with the Piggy Bac (PB) construct and the SuperPiggyBac transposase expression vector. Cas9 ribonucleoprotein particle (RNP) nucleofection was performed to obtain SUV39H1-KO cells as a control. On day 7, GFP-positive cells were sorted, and then the expression of SUV39H1 (western blot) and the memory marker CD27, as well as the trimethylation of H3K9 (FACS), were analyzed. The remaining cells were restimulated with transact on day 14 for new analysis.

[0191] In another approach, gene-activating CRISPR was used to upregulate AF196970.3 in cells as described below: Gene-activating CRISPR (Rankin et al., Overexpressing Long Noncoding RNAs Using Gene-activating CRISPR, Journal of Visualized Experiments (145), e59233 (2019) (Rankin et al., Overexpressing Long Noncoding RNAs Using Gene-activating CRISPR. J. Vis. Exp. (145), e59233 (2019).

[0192] These methods were used to overexpress AF196970.3 or portions thereof in human T cells. The levels of AF196970.3 were quantified, for example, by RT-PCR. The effects of AF196970.3 overexpression on 1) SUV39H1 protein levels (by western blot), 2) H3K9me3 levels (by FACS), 3) T cell phenotype (by FACS detecting CD27, CCR7, CD62L expression) were observed and compared to the inhibition of SUV39H1 by CRISPR knockout.

[0193] AF196970.3 overexpression inhibited SUV39H1 expression as measured by SUV39H1 protein levels, SUV39H1 activity levels, H3K9 trimethylation levels, or T cell phenotype (such as an increase in the memory phenotype as described in PCT / EP2020 / 070845). Briefly, to observe the expression of central memory T cell surface markers important for the memory phenotype of CD8+ T cells, T cells overexpressing AF196970.3 can be stimulated with aCD3+aCD28 beads for one week and then analyzed by flow cytometry. When Suv39h1 is silenced in T cells, the central memory T cell markers CCR7, CD27, and CD62L typically show increased expression levels. In addition, when Suv39h1 is silenced in T cells, the proportion of CCR7+CD45RO+CD27+CD62L+ cells that make up the central memory cell subset typically also increases. Generally, Suv39h1 silencing increases the proportion of central memory cells.

[0194] The effect of AF196970.3 overexpression on the efficacy of human CAR T cells in xenogeneic mouse models can also be compared to the inhibition of SUV39H1 by CRISPR KO. Example 4: Effect of lncRNA AF196970.3 inhibition on human T cells

[0195] The expression of AF196970.3 was suppressed to study its effect on human T cells. The gene was inactivated by CRISPR-Cas9 using gRNAs designed to target the first exon (SEQ ID NO: 2) or the promoter of AF196970.3.

[0196] The levels of AF196970.3 RNA (by RT-PCR) and SUV39H1 protein (by western blot) were quantified. The effects of AF196970.3 inhibition on 1) the level of H3K9me3 (by FACS) and 2) the T cell phenotype (by FACS detecting CD27, CCR7, CD62L expression) were observed.

[0197] Inhibition of AF196970.3 was predicted to increase the level of hSUV39H1. Example 5: Design of shRNA for silencing Suv39h1 gene and its overexpression in human cells

[0198] Due to the lack of inhibition of stemness- and memory-related genes, disruption of the SUV39H1 histone methyltransferase results in an enhanced T cell memory phenotype and in vivo persistence. Knockout can be used for adoptive T cell therapy and enables long-term enhancement of CAR-T cell activity, thereby improving survival in preclinical models. Based on this knowledge and the results obtained with lncRNAs shown in the previous examples, short hairpin RNAs (shRNAs) have been designed to silence Suv39h1 gene expression and inhibit or reduce the level and / or activity of SUV39H1 protein.

[0199] Figure 7 a shows a schematic diagram of the shRNA target sequence on the SUV39H1 gene. Figure 7 b shows the target sequences and loop sequences of different shRNAs (1 - 5) targeting SUV39H1. Figure 7 c shows a plasmid map of the lentiviral construct expressing shRNA in cells using the U6 promoter and the EGFP reporter gene. The experimental procedure was as Figure 8 shown in a. Briefly, HEK293 cells were seeded with 2.10 5 cells on day 0. On day 1, the cells were transduced with lentiviral particles at MOI = 5 and polybren (5 μg / mL), and removed 24 hours later. On day 4, the cells were split. On day 8, the cells were harvested and analyzed for GFP and SUV39H1 expression. Figure 8 b shows a comparison of GFP expression levels in untransduced cells with those in cells transduced with scrambled shRNA or one of the 5 shRNAs targeting SUV39H1 as determined by flow cytometry. As Figure 8As shown in c, Western blotting performed on day 8 showed that SUV39H1 staining in shRNA-transduced cells was significantly lower than actin staining compared to HEK293 controls and scrambled shRNA-transduced cells. Thus, when one of the shRNAs (1-5) disclosed herein was overexpressed, the level of Suv39H1 protein was significantly reduced. Example 6: Effect of Suv39h1 shRNA overexpression on human T cells

[0200] Figure 9 a depicts the experimental procedure for overexpressing shRNA (inhibiting Suv39h1 as described above) in CD8+ T cells. Briefly, T cell activation was performed on day 0. On day 1, cells were transduced with lentiviral particles at an MOI = 5 and polybren (4 μg / mL), and IL-2 was removed and added 24 hours later. On day 3, Cas9 ribonucleoprotein particles (RNPs) were nucleofected to obtain SUV39H1-KO cells as a control. On day 7, cells were harvested, and then the expression of SUV39H1 and the memory marker CD27, as well as trimethylation of H3K9, were analyzed.

[0201] Figure 9 b shows a comparison of GFP expression levels in untransduced cells with those in cells transduced with scrambled shRNA or one of five shRNAs targeting SUV39H1, as determined by flow cytometry. As Figure 9 c shown, Western blotting performed on day 7 showed that SUV39H1 staining in shRNA-transduced cells was significantly lower than actin staining compared to HEK293 controls and scrambled shRNA-transduced cells. Thus, when one of the shRNAs (1-5) disclosed herein was overexpressed, the level of Suv39H1 protein was significantly reduced. Consistently, Figure 9 d shows that in cells transduced with any one of the five shRNAs, the level of trimethylation of H3K9 was significantly reduced compared to cells transduced with scrambled shRNA and was similar to the level obtained in Suv39h1 KO cells. Figure 9 E to Figure 9 f further shows that cells transduced with shRNA exhibited an enhanced memory phenotype compared to cells transduced with scrambled shRNA, as shown by the increased CD27 expression on days 7 and 14.

[0202] All patents, patent applications, and publications are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were specifically and individually indicated to be incorporated by reference in its entirety for any and all purposes.

[0203] Although the foregoing articles and methods of the present disclosure have been described in terms of preferred embodiments and optional features, it will be apparent to those skilled in the art that variations or combinations can be applied without departing from the spirit and scope of the present disclosure. Such variations and combinations are intended to be within the meaning and scope of the present disclosure as defined by the claims. The breadth and scope of the present disclosure should not be limited by any of the above exemplary aspects, but should be defined only in accordance with the following claims and their equivalents. The terms and expressions used are used as descriptive terms and not as restrictive terms, and are not intended to exclude any equivalents of the features shown and described or portions thereof when such terms and expressions are used. 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Claims

1. A cell comprising a first nucleic acid, wherein the first nucleic acid is: (a) a heterologous nucleic acid that expresses an RNA comprising the nucleobase sequence of [SEQ ID NO: 1] (lncRNA AF196970.3) or a fragment thereof that is capable of inhibiting SUV39H1 expression in the cell; (b) a heterologous nucleic acid that comprises the nucleotide sequence of [SEQ ID NO: 5] (the genomic sequence encoding AF196970.3 (ENSG00000232828)) or a fragment thereof that expresses an RNA capable of inhibiting SUV39H1 expression in the cell; (c) a nucleic acid that expresses an RNA comprising the nucleobase sequence of [SEQ ID NO: 1] (lncRNA AF196970.3) or a fragment thereof that is capable of inhibiting SUV39H1 expression in the cell, the nucleic acid being operably linked to a heterologous expression control sequence (e.g., a promoter, enhancer, other regulatory sequences); or (d) a nucleic acid that is operably linked to a heterologous expression control sequence, comprises the nucleotide sequence of [SEQ ID NO: 5] (the genomic sequence encoding AF196970.3 (ENSG00000232828)) or a fragment thereof that expresses an RNA capable of inhibiting SUV39H1 expression in the cell, the nucleic acid being operably linked to a heterologous expression control sequence; (e) a heterologous nucleic acid that expresses any one of the nucleotide sequences of SEQ ID NOs: 13-17 and SEQ ID NOs: 26-30 or a fragment thereof that is capable of inhibiting SUV39H1 expression in the cell; (f) a heterologous nucleic acid that comprises the nucleotide sequences of SEQ ID NOs: 32-36 and SEQ ID NOs: 45-49 or a fragment thereof that expresses an RNA capable of inhibiting SUV39H1 expression in the cell; (g) a nucleic acid that expresses any one of SEQ ID NOs: 13-17 and SEQ ID NOs: 26-30 or a fragment thereof that is capable of inhibiting SUV39H1 expression in the cell, the nucleic acid being operably linked to a heterologous expression control sequence (e.g., a promoter, enhancer, other regulatory sequences); or (d) a nucleic acid that is operably linked to a heterologous expression control sequence, comprises the nucleotide sequences of SEQ ID NOs: 32-36 and SEQ ID NOs: 45-49 or a fragment thereof that expresses an RNA capable of inhibiting SUV39H1 expression in the cell, the nucleic acid being operably linked to a heterologous expression control sequence.

2. The cell according to claim 1, wherein, the first nucleic acid comprises the nucleotide sequence of any one of SEQ ID NOs: 5-9, 13-17 and 26-30 or a fragment thereof.

3. The cell according to claim 1, wherein, The first nucleic acid expresses RNA, comprising the nucleobase sequence of [SEQ ID NO: 2] (exon 1 of lncRNA AF196970.3) or any one of SEQ ID NOs: 13 - 17 and SEQ ID NOs: 26 - 30, or a fragment thereof capable of inhibiting the expression of SUV39H1 in the cell.

4. The cell according to claim 1 or 3, wherein, the first nucleic acid expresses RNA, comprising the nucleobase sequence of [SEQ ID NO: 3] (exon 2 of lncRNA AF196970.3) or a fragment thereof capable of inhibiting the expression of SUV39H1 in the cell.

5. The cell according to claim 1 or 3 or 4, wherein, the first nucleic acid expresses RNA, comprising the nucleobase sequence of [SEQ ID NO: 4] (exon 3 of lncRNA AF196970.3) or a fragment thereof capable of inhibiting the expression of SUV39H1 in the cell.

6. The cell according to any one of claims 1 to 5, wherein, the first nucleic acid expresses RNA having a length of at least about 12 - 50, 50 - 100, 100 - 150, 150 - 200, 200 - 250, 250 - 300, 300 - 350, 350 - 400, 400 - 450, 450 - 500, 500 - 550, 550 - 600, 600 - 650, 650 - 700, 700 - 750, 750 - 800, 800 - 850, 850 - 900, 250 - 750, 500 - 750 bases or more.

7. A cell, comprising: (a) a heterologous polynucleotide, comprising the nucleobase sequence of [SEQ ID NO: 1] (lncRNA AF196970.3) or the nucleotide sequence of any one of SEQ ID NOs: 13 - 17 and SEQ ID NOs: 26 - 30, or a fragment thereof capable of inhibiting the expression of SUV39H1 in the cell; or (b) a heterologous polynucleotide, comprising the nucleobase sequence of RNA expressed by [SEQ ID NO: 5] (genomic sequence) or any one of SEQ ID NOs: 32 - 36 and SEQ ID NOs: 45 - 49, or the nucleobase sequence of the RNA fragment capable of inhibiting the expression of SUV39H1 in the cell.

8. The cell according to claim 7, wherein, the polynucleotide has a length of at least about 12 - 50, 50 - 100, 100 - 150, 150 - 200, 200 - 250, 250 - 300, 300 - 350, 350 - 400, 400 - 450, 450 - 500, 500 - 550, 550 - 600, 600 - 650, 650 - 700, 700 - 750, 750 - 800, 800 - 850, 850 - 900, 250 - 750, 500 - 750 bases or more.

9. The cell according to any one of the preceding claims, wherein, The cell is a modified immune cell.

10. The cell according to any one of the preceding claims, wherein, the cell is a T cell, CD4+ T cell, CD8+ T cell, CD4+ and CD8+ T cell, NK cell, Treg cell, Tm cell, memory stem T cell (TSCM), TCM cell, TEM cell, monocyte, dendritic cell, macrophage, T cell progenitor, NK cell progenitor, pluripotent stem cell, induced pluripotent stem cell (iPSC), hematopoietic stem cell (HSC), adipose stem cell (ADSC), myeloid or lymphoid multipotent stem cell.

11. The cell according to any one of the preceding claims, further comprising one or more engineered receptors, two or more engineered receptors, or three or more engineered receptors.

12. The cell according to any one of the preceding claims, further comprising a second heterologous nucleic acid expressing one or more engineered receptors.

13. The cell according to claim 11 or 12, wherein, the engineered receptor comprises: a) an extracellular antigen-binding domain that specifically binds an antigen, optionally comprising an antibody heavy chain variable region and / or an antibody light chain variable region, and optionally being bispecific or trispecific; b) a transmembrane domain, optionally comprising a transmembrane domain fragment of the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD34, CD137, or CD154, NKG2D, OX40, ICOS, 2B4, DAP10, DAP12, CD40; and c) optionally one or more co-stimulatory domains from 4-1BB, CD28, ICOS, OX40, DAP10, or DAP12, 2B4, CD40, FCER1G; d) An intracellular signaling domain, comprising an intracellular signaling domain or any fragment thereof from CD3ζ, FcRγ 、 FcRβ, CD3γ, CD3δ, CD3ε, CDS, CD22, CD79a, CD79b or CD66d, 2B4.

14. The cell according to claim 13, wherein, the engineered receptor is a chimeric antigen receptor (CAR) comprising: a) an extracellular antigen-binding domain, optionally an scFv, b) a transmembrane domain, optionally from CD28, CD8, or CD3ζ, c) one or more co-stimulatory domains, optionally from 4-1BB, CD28, ICOS, OX40, or DAP10, and d) an intracellular signaling domain from CD3ζ, optionally wherein ITAM2 and ITAM3 have been inactivated.

15. The cell according to claim 13, wherein, the engineered receptor is a modified TCR comprising: a) a first antigen-binding chain comprising an antigen-binding fragment of an antibody heavy chain variable region (VH); and b) a second antigen-binding chain comprising an antigen-binding fragment of an antibody light chain variable region (VL); Wherein the first antigen-binding chain and the second antigen-binding chain each comprise a TRAC polypeptide or a TRBC polypeptide, optionally wherein at least one of the TRAC polypeptide and the TRBC polypeptide is endogenous, and optionally wherein one or both of the endogenous TRAC polypeptide and the TRBC polypeptide are inactivated.

16. The cell according to claim 13, wherein, the engineered receptor is a chimeric co-stimulatory receptor comprising (a) an extracellular domain of a co-stimulatory ligand, optionally from CD80, (b) a transmembrane domain, optionally from CD80, and (c) an intracellular domain of a co-stimulatory molecule, optionally CD28, 4-1BB, OX40, ICOS, DAP10, CD27, CD40, NKGD2 or CD2, preferably 4-1BB.

17. The cell according to any one of claims 13 to 15, wherein, The extracellular antigen-binding domain binds the antigen with a KD affinity of about 1 x 10 -7 or lower, about 5 x 10 -8 or lower, about 1 x 10 -8 or lower, about 5 x 10 -9 or lower, about 1 x 10 -9 or lower, about 5 x 10 -10 or lower, about 1 x 10 -10 or lower, about 5 x 10 -11 or lower, about 1 x 10 -11 or lower, about 5 x10 -12 or lower, or about 1 x 10 -12 or lower.

18. The cell according to any one of claims 13 to 15, wherein, the antigen has a low density on the cell surface, less than about 10,000 molecules per cell, or less than about 5,000 molecules, or less than about 2,000 molecules.

19. The cell according to any one of claims 13 to 18, wherein, the extracellular antigen-binding domain binds to orphan tyrosine kinase receptor ROR1, tEGFR, Her2, p95HER2, LI-CAM, CD19, CD20, CD22, mesothelin, CEA, Claudin 18.2, hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, CD70, EGFR, EGP-2, EGP-4, EphA2, ErbB2, ErbB3 or ErbB4, FBP, FcRH5, fetal acetylcholine e receptor, GD2, GD3, HMW-MAA, IL-22R-α, IL-13R-α2, kdr, κ light chain, BCMA, Lewis Y, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D ligand, NY-ESO-1, MART-1, gp100, oncofetal antigen, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate-specific antigen (PSMA), estrogen receptor, progesterone receptor, ephrinB2, CD 123, CS-1, c-Met, GD-2, MAGE A3, CE7 or Wilms tumor 1 (WT-1), or optionally, the extracellular antigen-binding domain binds to any tumor neoantigen peptide disclosed in International Patent Publication WO 2021 / 043804.

20. The cell according to any one of the preceding claims, comprising two engineered antigen receptors, each receptor binding a different antigen.

21. The cell according to any one of the preceding claims, which is autologous or allogeneic.

22. The cell according to any one of the preceding claims, wherein, The expression of SUV39H1 is reduced or inhibited by at least about 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95%.

23. The cell according to any one of the preceding claims, wherein, the cell is isolated from a subject having cancer or at risk of developing cancer.

24. A modified oligonucleotide comprising a nucleobase sequence from any one of [SEQ ID NO: 1-4] that is at least about 12 bases in length, wherein the modified oligonucleotide comprises one or more of a modified backbone linkage, a modified sugar moiety, a modified phosphate moiety, a modified nucleobase or a chemical conjugate moiety.

25. The modified oligonucleotide according to claim 24, which has a length of at least about 12-50, 50-75 or 50-100 bases.

26. The modified oligonucleotide according to claims 24 to 25, wherein, the modified backbone linkage comprises phosphorothioate, phosphonoacetate, thiophosphonoacetate, methylphosphonate, boranophosphate or dithiophosphate.

27. The modified oligonucleotide according to claims 24 to 26, wherein, the modified sugar is modified to replace the 2’OH-group with another group, which is optionally H, -OR, -R (where R can be, for example, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), halogen, -F, -Br, -Cl or -I, -SH, -SR (where R can be, for example, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), -arabinose, F-arabinose, amino (where amino can be, for example, NH2; alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylamino, diheteroarylamino or amino acid); or cyano (-CN); optionally 2’-O-methoxy, 2’-O-methoxyethyl modification, 2’-fluoro, 2’-deoxy or a combination thereof.

28. The modified oligonucleotide according to claims 24 to 27, wherein, the modified nucleobase comprises one or more of 5-methylcytosine, modified uridines such as 5-(2-amino)propyl uridine and 5-bromouridine, modified adenosines and guanosines such as modified at position 8, such as 8-bromoguanosine, deazapurines such as 7-deazaadenosine, or O- and N-alkylated nucleotides such as N6-methyladenosine, or polycyclic modified nucleotides (e.g., tricyclic; and "unlocked" forms such as glycol nucleic acid (GNA) (e.g., R-GNA or S-GNA, where ribose is replaced by a glycol unit linked to a phosphodiester bond), or threose nucleic acid (TNA, where ribose is replaced by α-L-threofuranosyl-(3’→2’)).

29. A nucleic acid operably linked to a heterologous expression control sequence, said nucleic acid comprising (a) a nucleotide sequence encoding or expressing an RNA, comprising the nucleobase sequence of [SEQ ID NO: 1] (lncRNA AF196970.3) or the nucleotide sequence of any one of SEQ ID NOs: 13 - 17 and SEQ ID NOs: 26 - 30 or a fragment thereof capable of inhibiting the expression of SUV39H1 in said cell, or (b) the nucleotide sequence of [SEQ ID NO: 5] (genomic sequence encoding AF196970.3 (ENSG00000232828)) or the nucleotide sequence of any one of SEQ ID NOs: 32 - 36 and SEQ ID NOs: 45 - 49 or a fragment thereof expressing an RNA capable of inhibiting the expression of SUV39H1 in said cell.

30. The nucleic acid according to claim 29, which comprises the nucleotide sequence of any one of SEQ ID NOs: 5 - 9, 13 - 17, 26 - 30, 32 - 36, 45 - 49 or one or more fragments thereof.

31. The nucleic acid according to any one of claims 29 to 30, wherein, the heterologous control sequence is a constitutive, inducible or tissue - specific promoter, optionally EF1α, CMV, SFFV, hPGK, RPBSA or CAG.

32. A vector comprising the nucleic acid according to any one of claims 29 to 30 and one or more additional expression control sequences.

33. The vector according to claim 32, which is a viral vector, optionally an adenovirus, adeno - associated virus (AAV), poxvirus, papillomavirus, lentivirus, retrovirus, herpesvirus, foamy virus or Semliki Forest virus vector, and includes pseudotyped viruses.

34. The nucleic acid or vector according to any one of claims 29 to 33, which is in a delivery vehicle, said delivery vehicle optionally being a liposome, lipid - containing complex, nanoparticle, gold particle or polymer complex.

35. A method of preparing a cell according to any one of claims 1 to 23, comprising (a) introducing (i) the nucleic acid according to any one of claims 28 to 30 or 33 or (ii) the vector according to any one of claims 30 to 33 into said cell, and optionally (b) introducing a nucleic acid encoding an antigen - specific receptor into said cell.

36. A method for preparing a cell according to any one of claims 1 to 23, comprising introducing a heterologous expression control sequence into the cell in such a way that it is operably linked to an endogenous nucleic acid that expresses RNA, the endogenous nucleic acid comprising the nucleobase sequence of [SEQ ID NO: 1] (lncRNA AF196970.3), or the nucleotide sequence of any one of SEQ ID NOs: 13 - 17 and SEQ ID NOs: 26 - 30 or a fragment thereof capable of inhibiting the expression of SUV39H1 in the cell, optionally a nucleic acid comprising the nucleotide sequence of [SEQ ID NO: 5] (genomic sequence encoding AF196970.3 (ENSG00000232828)) or the nucleotide sequence of any one of SEQ ID NOs: 32 - 36 and SEQ ID NOs: 45 - 49 or an allelic variant thereof.

37. A method for treating a disease using a cell according to any one of claims 1 to 23, comprising administering to a subject in need thereof an effective amount of the cell for treating the disease, optionally cancer, infectious disease, autoimmune disease, inflammatory disease or allergic disease, wherein the cell expresses one or more antigen - specific receptors that bind to antigens associated with the disease.

38. A method for treating a subject suffering from cancer, comprising administering to the subject: (1) a cell according to any one of claims 1 to 23, or a modified oligonucleotide according to any one of claims 24 to 28, or a nucleic acid according to any one of claims 29 to 31, or a vector according to any one of claims 31 to 33, and (2) a second cancer therapeutic agent.

39. The method according to claim 38, wherein, the second cancer therapeutic agent is an immune checkpoint modulator, cancer vaccine, chemotherapeutic agent or anti - angiogenic agent.

40. A method for treating a subject suffering from cancer, comprising administering to the subject: (1) a cell according to any one of claims 1 to 23, wherein the cell is a T cell, NK cell, lymphoid progenitor cell or myeloid progenitor cell, comprising a genetically engineered antigen receptor, wherein the expression of the SUV39H1 gene is inhibited, and wherein the inhibition of the SUV39H1 gene results in enhanced anti - cancer activity of the immune cell; and (2) an immune checkpoint modulator.

41. The method according to claim 40, wherein, the immune checkpoint modulator is an inhibitor of PD1, CTLA4, LAG3, BTLA, OX2R, TIM - 3, TIGIT, LAIR - 1, PGE2 receptor, EP2 / 4 adenosine receptor or A2AR.

42. The method according to claim 41, wherein, the immune checkpoint modulator is an anti - PD - 1 inhibitor or an anti - PDL - 1 inhibitor.

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